Compositions and methods for preparation of poorly water soluble drugs with increased stability

ABSTRACT

The present invention provides stable pharmaceutical compositions of poorly water soluble pharmaceutical agents and stabilizing agents which function to increase stability of the compositions. The use of stabilizing agents provide extended stability of nanoparticle suspensions and other formulations of poorly water soluble pharmaceutical agents such as docetaxel under certain conditions, for example upon dilution for administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication 60/712,865 filed Aug. 31, 2005, U.S. Provisional Application60/736,962 filed Nov. 14, 2005, and U.S. Provisional Application60/736,931 filed Nov. 14, 2005, all of which are hereby incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

There is an ever increasing number of pharmaceutical drugs beingformulated that are poorly soluble or insoluble in aqueous solutions.Such drugs provide challenges to delivering them in an injectable formsuch as through parenteral administration. A well-designed formulationmust, at a minimum, be capable of presenting a therapeutically effectiveamount of the poorly soluble drug to the desired absorption site, in anabsorbable form. In addition, these compositions tend to be unstable,with sedimentation and/or precipitation occurring in under 24 hoursfollowing rehydration or reconstitution.

Taxanes, in particular the two currently available taxane drugs,paclitaxel and docetaxel, are potent antitumor agents. Paclitaxel isvery poorly water soluble (less than 10 μg/mL), and as a result, cannotbe practically formulated with an aqueous medium for IV administration.Currently, paclitaxel is formulated for IV administration to patientswith cancer in a solution with polyoxyethylated castor oil (Polyoxyl 35or Cremophor®) as the primary solvent/surfactant, with highconcentrations of ethanol employed as co-solvent. One of the majordifficulties in the administration of paclitaxel is the occurrence ofhypersensitivity reactions. These reactions, which include severe skinrashes, hives, flushing, dyspnea, tachycardia and others, may beattributed at least in part to the high concentrations of ethanol andCremophor used as solvents in the formulation. Docetaxel, an analog ofpaclitaxel, is semisynthetically produced from 10-deacetyl baccatin III,a noncytotoxic precursor extracted from the needles of Taxus baccata andesterified with a chemically synthesized side chain (Cortes and Pazdur,1995, J. Clin. Oncol. 13(10):2643-55). Like paclitaxel, docetaxel isvery poorly soluble in water. Currently, the most preferredsolvent/surfactant used to dissolve docetaxel is polysorbate 80 (Tween80) (Bissery et al. 1991 Cancer Res. 51(18):4845-52; Tomiak et al.1992). Like Cremophor, Tween often causes hypersensitivity reactions inpatients. Further, Tween 80 cannot be used with PVC delivery apparatusbecause of its tendency to leach diethylhexyl phthalate, which is highlytoxic.

Purification of semi-synthetic paclitaxel and docetaxel is a challengingproblem due to the formation of a number of degradation products alongthe synthetic route. Furthermore, purified taxanes are found to undergodegradation, even under controlled storage conditions. Therefore, itbecomes desirable to develop stable forms of these molecules whichretain the desirable anti-cancer properties. Previous efforts inobtaining suitable docetaxel have been focusing on processes ofpreparing trihydrate forms of docetaxel, which were believed to havesubstantially greater stability than that of the anhydrous product. See,e.g., U.S. Pat. Nos. 6,022,985; 6,838,569.

In order to attain the expected therapeutic effects of poorly watersoluble agents such as paclitaxel and docetaxel, it is usually requiredthat a solubilized form or nanodispersed form of the agent beadministered to a patient.

Thus, a number of methods have been developed which are based on the useof auxiliary solvents; surfactants; soluble forms of the drug, e.g.,salts and solvates; chemically modified forms of the drug, e.g.,prodrugs; soluble polymer-drug complexes; special drug carriers such asliposomes; and others. Indeed, the use of amphiphilic block copolymermicelles has attracted a great deal of interest as a potentiallyeffective drug carrier which is capable of solubilizing a hydrophobicdrug in an aqueous environment.

Each of the above methods is hampered by one or more particularproblems. For example, the method based on the use of surfactantmicelles to solubilize hydrophobic drugs has problems in that some ofthe surfactants are relatively toxic and precipitation of hydrophobicdrugs occurs when subjected to dilution.

Previously, phospholipid-based liposome formulations for paclitaxel,Taxotere, and other active taxanes have been developed (Straubinger etal. 1993, J. Natl. Cancer Inst. Monogr. (15):69-78; Straubinger et al1994; Sharma et al. 1993, Cancer Res. 53(24):557-81; Sharma andStraubinger 1994, Pharm. Res. 11(6):889-96; A. Sharma et al. 1995, J.Pharm. Sci. 84(12):1400-4), and the physical properties of these andother taxane formulations have been studied (Sharma and Straubinger1994, Pharm. Res. 11(6):889-96; U. S. Sharma et al. 1995, J. Pharm. Sci.84(10):1223-30; Balasubramanian and Straubinger 1994, Biochemistry33(30):8941-7; Balasubramanian et al. 1994, J. Pharm. Sci.83(10):1470-6). The main utility of these formulations is theelimination of toxicity related to the Cremophor EL excipient, and areduction in the toxicity of the taxane itself, as demonstrated inseveral animal tumor models (Sharma et al. 1993, Cancer Res.53(24):557-81; A. Sharma et al. 1995, J. Pharm. Sci. 84(12):1400-4;Sharma et al. 1996, Cancer Lett. 107(2):265-272). This observation holdsfor several taxanes in addition to paclitaxel (A. Sharma et al. 1995, J.Pharm. Sci. 84(12):1400-4). In some cases, the antitumor potency of thedrug appears to be slightly greater for the liposome-based formulations(Sharma et al. 1993, Cancer. Res. 53(24):557-81).

These liposomal formulations comprise phospholipids and other additives,in addition to the taxane, and may be stored in a dried state. Uponaddition of an aqueous phase to the mixture, particles formspontaneously and may take the form of liposomes (Straubinger et al.1993). Liposomes are closed, vesicular structures consisting of alimiting bilayer membrane surrounding an aqueous core. A preferredformulation composition (Sharma and Straubinger 1994) contains a neutral(zwitterionic) phospholipid such as lecithin (phosphatidylcholine,80-90% by mole ratio), along with a negatively charged phospholipid suchas phosphatidylglycerol (10-20%). The latter prevents aggregation of theparticles through electrostatic repulsion. The most stable taxanecontent is in the range of 3-4 mole % (relative to total phospholipidcontent); such Liposomes may be physically and/or chemically stable for2 months after hydration. Under most conditions, paclitaxel formulationscontaining higher (e.g. 8 mole %) drug concentrations are very unstableand may precipitate within minutes of preparation (Sharma andStraubinger 1994).

The greatest concern over these formulations has been the relatively lowtaxane content of acceptably stable formulations (3-5 mole %), whichnecessitates the administration of a large amount of phospholipid (5-10gm) to patients in order to give the anticipated dose of drug. Althoughhumans frequently are given large amounts of lipids intravenously forTotal Parenteral Nutrition (TPN), a major developmental aim has been toproduce taxane liposomes having a higher taxane content.

Other approaches to formulating poorly soluble drug for oral orparenteral delivery include, for example, formulations in which thepoorly soluble drug is an oil-in-water emulsion, a microemulsion, or asolution of micelles or other multi-lamellar carrier particles. Whilesuch approaches may be appropriate for some ionizable as well asnon-ionizable hydrophobic therapeutic agents, they fail to takeadvantage of the unique acid-base chemical properties, and associatedsolubility properties, of ionizable compounds.

Drugs that are insoluble in water can have significant benefits whenformulated as a stable suspension of sub-micron particles. Accuratecontrol of particle size is essential for safe and efficacious use ofthese formulations. Particles must be less than seven microns indiameter to safely pass through capillaries without causing emboli(Allen et al., 1987; Davis and Taube, 1978; Schroeder et al., 1978;Yokel et al., 1981, Toxicol. Lett. 9(2):165-70).

Another approach is disclosed in U.S. Pat. No. 5,118,528 which disclosesa process for preparing nanoparticles. The process includes the stepsof: (1) preparing a liquid phase of a substance in a solvent or amixture of solvents to which may be added one or more surfactants, (2)preparing a second liquid phase of a non-solvent or a mixture ofnon-solvents, the non-solvent being miscible with the solvent or mixtureof solvents for the substance, (3) adding together the solutions of (1)and (2) with stirring and (4) removing of unwanted solvents to produce acolloidal suspension of nanoparticles. The '528 patent discloses that itproduces particles of the substance smaller than 500 nm without thesupply of energy. In particular the '528 patent states that it isundesirable to use high energy equipment such as sonicators andhomogenizers.

U.S. Pat. No. 4,826,689 discloses a method for making uniformly sizedparticles from water-insoluble drugs or other organic compounds. First,a suitable solid organic compound is dissolved in an organic solvent,and the solution can be diluted with a non-solvent. Then; an aqueousprecipitating liquid is infused, precipitating non-aggregated particleswith substantially uniform mean diameter. The particles are thenseparated from the organic solvent. Depending on the organic compoundand the desired particle size, the parameters of temperature, ratio ofnon-solvent to organic solvent, infusion rate, stir rate, and volume canbe varied according to the patent. The '689 patent discloses that thisprocess forms a drug in a metastable state which is thermodynamicallyunstable and which eventually converts to a more stable crystallinestate. The '689 patent discloses trapping the drug in a metastable statein which the free energy lies between that of the starting drug solutionand the stable crystalline form. The '689 patent discloses utilizingcrystallization inhibitors (e.g., polyvinylpyrrolidinone) andsurface-active agents (e.g., poly(oxyethylene-co-oxypropylene)) torender the precipitate stable enough to be isolated by centrifugation,membrane filtration or reverse osmosis.

Another approach to providing insoluble drugs for parenteral delivery isdisclosed in U.S. Pat. No. 5,145,684. The '684 patent discloses the wetmilling of an insoluble drug in the presence of a surface modifier toprovide a drug particle having an average effective particle size ofless than 400 nm. The '684 patent discloses the surface modifier isadsorbed on the surface of the drug particle in an amount sufficient toprevent agglomeration into larger particles. Nanoparticles of insolubledrugs prepared under conditions of high shear forces (e.g., sonication,high pressure homogenization, or the like) with biocompatible polymers(e.g., albumin) are disclosed in, for example, U.S. Pat. Nos. 5,916,596,6,506,405, and 6,537,579 and also in U.S. Patent Publication2005/0004002 A1

In view of the foregoing, there is a need for pharmaceuticalcompositions comprising poorly water soluble drugs with increasedphysical and chemical stability, which eliminate the use ofphysiologically harmful solvents and excipients, and methods ofproduction thereof. It is desirable that such pharmaceuticalcompositions should not degrade, should remain stable under storageconditions and remain physically and/or chemically stable afterrehydration. It would also be desirable to have a pharmaceuticalcomposition comprising an anhydrous form of poorly water soluble drugthat has greater solubility in traditionally used solvents andexcipients, as well as in solvents and excipients that are notphysiologically harmful. The present invention provides suchpharmaceutical compositions and methods.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entireties.

BRIEF SUMMARY OF THE INVENTION

The invention provides compositions and methods of producing stablepharmaceutical formulations of docetaxel. In one embodiment, theinvention provides pharmaceutical formulations of docetaxel comprisingcitrate or derivatives thereof. In a second embodiment, the inventionprovides pharmaceutical formulations of docetaxel comprising sodiumpyrophosphate. In a third embodiment, the invention providespharmaceutical formulations of docetaxel comprising EDTA or derivativethereof. In a fourth embodiment, the invention provides pharmaceuticalformulations of docetaxel comprising sodium gluconate. In a fifthembodiment, the invention provides pharmaceutical formulations ofdocetaxel comprising citrate and sodium chloride. In a sixth embodiment,the invention provides a formulation of docetaxel comprising asurfactant, wherein the docetaxel used for preparing the formulation isin an anhydrous form prior to being incorporated into the formulation.

Accordingly, in one aspect, the invention provides compositions (such aspharmaceutical compositions) comprising a poorly water solublepharmaceutical agent (such as docetaxel) and a stabilizing agent,wherein stability of the composition is enhanced as compared to that ofa composition without the stabilizing agent. In some embodiments, thecompositions further comprise a biocompatible polymer (such as carrierproteins described herein). The stabilizing agent includes, for example,chelating agents (such as citrate, malic acid, edetate, and pentetate),sodium pyrophosphate, and sodium gluconate.

In another aspect, there are provided various compositions (such aspharmaceutical compositions), comprising docetaxel, wherein thedocetaxel used for preparation of the composition is in anhydrous form(for example, the docetaxel may be anhydrous prior to being incorporatedinto the composition). In some embodiments the composition furthercomprises a biocompatible polymer (such as a carrier protein describedherein). In some embodiments, the composition further comprises astabilizing agent (such as stabilizing agents described herein). In someembodiments, the composition comprises both a biocompatible polymer(such as carrier proteins described herein) and a stabilizing agent. Insome embodiments, the invention provides compositions (such aspharmaceutical compositions) comprising docetaxel and a surfactant,wherein the docetaxel used for preparation of the composition is inanhydrous form (for example, the docetaxel may be anhydrous prior tobeing incorporated into the composition). In some embodiments, thecomposition further comprises a stabilizing agent (such as stabilizingagents described herein).

Also provided are unit dosage forms of compositions described herein,articles of manufacture comprising the inventive compositions or unitdosage forms in suitable packaging, and kits comprising thecompositions. The invention also provides methods of making and usingthese compositions as described herein.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows body weight loss of rats at 5 mg/kg docetaxel dose for ananoparticle albumin formulation of docetaxel (Nab-docetaxel) and Tween80-docetaxel (Taxotere®). Dosing occurred on days 0, 4, and 8.

FIG. 2 shows neutropenia comparison in rats at 5 mg/kg dose forNab-docetaxel and Tween 80-docetaxel (Taxotere). Dosing occurred on days0, 4, and 8.

FIGS. 3A-3D show the pharmacokinetic comparison of Nab-docetaxel andTaxotere. FIGS. 3A-3C show plasma concentration of Nab-docetaxel andTaxotere® at 10 mg/kg, 20 mg/kg, and 30 mg/kg doses, respectively. FIG.3D shows the linear relationship between AUC (Area Under Curve) and dosefor Nab-docetaxel and nonlinear relationship between AUC and dose forTaxotere. Nab-docetaxel exhibited a linear relationship fitted by theequation AUC=218*Dose; Taxotere exhibited an exponential curve fitted bythe equation AUC=722*exp(0.10*Dose).

FIG. 4 shows the inhibition of drug binding to albumin in the presenceof surfactant Tween 80 and Cremophor EL®/EtOH.

FIGS. 5A and 5B show antitumor activity (5A) and body weight loss (5B)with Nab-docetaxel in a H29 colon tumor xenograft mice. Mice were dosedwith Nab-docetaxel at 15 mg/kg, q4dx3.

FIGS. 6A and 6B show antitumor activity (6A) and body weight loss (6B)in a HCT116 colon tumor xenograft mouse dosed with saline, Nab-docetaxel(22 mg/kg), and Taxotere (15 mg/kg).

FIGS. 7A and 7B show body weight loss (7A) and antitumor activity (7B)in a PC3 prostate tumor xenograft mouse dosed with saline, Nab-docetaxel(10, 15, 20, 30 mg/kg), and Tween 80-docetaxel (10 mg/kg).

DETAILED DESCRIPTION OF THE INVENTION

The present invention in one of its embodiments provides forcompositions and methods of preparation of docetaxel and other poorlywater soluble pharmaceutical agents or drugs which retain the desirabletherapeutic effects and remain physically and/or chemically stable uponexposure to certain conditions such as prolonged storage, elevatedtemperature, or dilution for parenteral administration.

A stable composition is, for example, one that remains physically and/orchemically stable and therefore does not show evidence of precipitationor sedimentation for at least about 8 hours, including for example atleast about any of 24 hours, 48 hours, or up to about 96 hours followingreconstitution or rehydration. For example, the compositions may remainstable for at least 24 hours following reconstitution or rehydration.

Stability of a suspension is generally (but not necessarily) evaluatedat usual conditions of transport and storage expected during productdistribution (such as room temperature (such as 20-25° C.) orrefrigerated conditions (such as 4° C.)). For example, a suspension isstable at a storage temperature if it exhibits no flocculation orparticle agglomeration visible to the naked eye or when viewed under theoptical microscope at 1000 times (or other suitable particlecharacterization techniques), at about fifteen minutes after preparationof the suspension. Stability may also be evaluated under exaggeratedconditions of temperature, humidity, light, and/or others, to test thestability of the compositions in an accelerated testing. For example,stability can be evaluated at a temperature that is higher than about40° C. Stability of the composition can also be evaluated, for example,by the ability of the composition to remain suspended without showingevidence of settling or creaming, or by the ability of the compositionto remain unchanged (i.e., no visible difference) in terms of color orconsistency.

Stability of a dry (such as a lyophilized) composition can be evaluatedbased on the behavior of the liquid suspension resulting fromreconstitution or rehydration of the dry composition.

It is an object of the invention to provide pharmaceutical compositionscapable of maintaining physically and/or chemically stabilized,therapeutically effective amounts of poorly water soluble pharmaceuticalagents. It is another object of the invention to provide pharmaceuticalcompositions capable of maintaining a physically and/or chemicallystabilized poorly water soluble pharmaceutical agents upon dilution foradministration to a patient. It is a further object of the invention toprovide pharmaceutical compositions capable of maintaining physicallyand/or chemically stabilized, therapeutically effective amounts ofpoorly water soluble pharmaceutical agents with reduced toxicities. Itis a further object of the invention to provide stable pharmaceuticalformulations using anhydrous docetaxel, as well compositions resultingfrom use of anhydrous docetaxel.

It is a further object of the invention to provide improved methods ofpreparing pharmaceutical compositions capable of maintaining physicallyand/or chemically stabilized, therapeutically effective amounts ofpoorly water soluble pharmaceutical agents. It is a further object ofthe invention to provide improved methods of preparing pharmaceuticalcompositions capable of maintaining a physically and/or chemicallystabilized poorly water soluble pharmaceutical agent upon dilution foradministration to a patient. It is a further object of the invention toprovide improved methods of preparing pharmaceutical compositionscapable of maintaining physically and/or chemically stabilized,therapeutically effective amounts of poorly water soluble pharmaceuticalagents with reduced toxicities.

In one embodiment the invention provides a sterile pharmaceuticalcomposition for parenteral administration comprised of a poorly watersoluble pharmaceutical agent, which is physically and/or chemicallystabilized by the addition of excipients to the composition. Prior tothe present invention, the relative stability of certain poorly solublepharmaceutical agents has limited their use in parenteral pharmaceuticalcompositions due to degradation under storage conditions and/orprecipitation upon dilution. Many different pharmaceutical agents couldnot be satisfactorily prepared as parenterals due to the absence of astable composition.

The present invention involves the surprising discovery that commonexcipients such as citrate are capable of stabilizing poorly watersoluble pharmaceutical agents such as docetaxel. It is therefore aprimary object of the invention to provide compositions comprisingdocetaxel (and other poorly water soluble pharmaceutical agents) andexcipients to obtain stable, parenteral pharmaceutical compositions.Therefore, in one embodiment, the invention provides a pharmaceuticalcomposition comprising docetaxel and citrate. In another embodiment, theinvention provides a pharmaceutical composition comprising docetaxel,citrate and sodium chloride.

Various Embodiments of the Invention

The invention provides compositions (such as pharmaceuticalcompositions) comprising a poorly water soluble pharmaceutical agent anda stabilizing agent, wherein stability of the composition is enhanced ascompared to that of a composition without the stabilizing agent. Forexample, the composition may comprise docetaxel and a stabilizing agent,wherein stability of the composition is enhanced as compared to that ofa composition without the stabilizing agent. In some embodiments, thecompositions further comprise a biocompatible polymer. In someembodiments, the biocompatible polymer is a carrier protein (such as analbumin, for example, human serum albumin (HSA)). In some embodiments,the stability of the composition is at least 1.5 times (including forexample at least about any of 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×,20×, 25×, 30×, or more) greater as compared to that of a compositionwithout the stabilizing agent in some embodiments, the poorly watersoluble pharmaceutical agent is unstable in a composition not comprisingthe stabilizing agent.

In some embodiments, there is provided a composition comprising a poorlywater soluble pharmaceutical agent and a stabilizing agent, wherein thestabilizing agent is a chelating agent, and wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, there is provided a compositioncomprising docetaxel and a stabilizing agent, wherein the stabilizingagent is a chelating agent, and wherein stability of the composition isenhanced as compared to that of a composition without the stabilizingagent. In some embodiments, the composition further comprises abiocompatible polymer. In some embodiments, the biocompatible polymer isa carrier protein (such as albumin, for example, HSA). In someembodiments, the stabilizing agent is a polydentate chelating agent. Insome embodiments, the stabilizing agent comprises one or more carboxylicacid groups. In some embodiments, the chelating agent is notdeferoxamine (i.e., is other than deferoxamine). In some embodiments,the chelating agent is any of (and in some embodiments selected from thegroup consisting of) edetate, citrate, malic acid, pentetate,tromethamine, derivatives thereof, and mixtures thereof. In someembodiments, the stabilizing agent is a citrate or a derivative thereof(such as sodium citrate and in some embodiments citric acid). In someembodiments, the composition comprises sodium citrate and sodiumchloride. In some embodiments, the composition comprises about 200 mMcitrate and about 300 mM sodium chloride. In some embodiments, thestabilizing agent is an edetate or a derivative thereof (such as EDTA).

In some embodiments, there is provided a composition comprising a poorlywater soluble pharmaceutical agent and a stabilizing agent, wherein thestabilizing agent is sodium gluconate, and wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, there is provided a compositioncomprising docetaxel and a stabilizing agent, wherein the stabilizingagent is sodium gluconate, and wherein stability of the composition isenhanced as compared to that of a composition without the stabilizingagent. In some embodiments, the composition further comprises abiocompatible polymer. In some embodiments, the biocompatible polymer isa carrier protein (such as albumin, for example, HSA).

In some embodiments, there is provided a composition comprising a poorlywater soluble pharmaceutical agent and a stabilizing agent, wherein thestabilizing agent is sodium pyrophosphate, and wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, there is provided a compositioncomprising docetaxel and a stabilizing agent, wherein the stabilizingagent is sodium pyrophosphate, and wherein stability of the compositionis enhanced as compared to that of a composition without the stabilizingagent. In some embodiments, the composition further comprises abiocompatible polymer. In some embodiments, the biocompatible polymer isa carrier protein (such as albumin, for example, HSA).

In some embodiments, the composition comprises a poorly water solublepharmaceutical agent, an albumin, and a stabilizing agent, wherein theweight ratio of the albumin to the poorly water soluble pharmaceuticalagent in the composition is about 0.01:1 to about 100:1, and whereinstability of the composition is enhanced as compared to that of acomposition without the stabilizing agent. In some embodiments, thecomposition comprises a poorly water soluble pharmaceutical agent, analbumin, and a stabilizing agent, wherein the weight ratio of thealbumin to the poorly water soluble pharmaceutical agent in thecomposition is about 18:1 or less (including for example any of about1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 12:1,about 4:1 to about 10:1, and about 9:1), and wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, the composition comprisesdocetaxel, an albumin, and a stabilizing agent, wherein the weight ratioof the albumin to the docetaxel in the composition is about 18:1 orless. (including for example any of about 1:1 to about 18:1, about 2:1to about 15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, andabout 9:1), and wherein stability of the composition is enhanced ascompared to that of a composition without the stabilizing agent. In someembodiments, the stabilizing agent is a chelating agent, such as any of(and in some embodiments selected from the group consisting of) edetate,citrate, malic acid, pentetate, tromethamine, derivatives thereof, andmixtures thereof. In some embodiments, the stabilizing agent is acitrate or a derivative thereof (such as sodium citrate). In someembodiments, the composition comprises sodium citrate and sodiumchloride. In some embodiments, the stabilizing agent is an edetate or aderivative thereof (such as EDTA). In some embodiments, the stabilizingagent is sodium gluconate. In some embodiments, the stabilizing agent issodium pyrophosphate.

In some embodiments, the protein/pharmaceutical agent is in particulateform(s), which in various embodiments may be of average diameters asdescribed herein.

In some embodiments, the composition comprises a protein-associatedpoorly water soluble pharmaceutical agent and a stabilizing agent,wherein stability of the composition is enhanced as compared to that ofa composition without the stabilizing agent. In some embodiments, thecomposition comprises a protein-associated docetaxel and a stabilizingagent, wherein stability of the composition is enhanced as compared tothat of a composition without the stabilizing agent. In someembodiments, the stabilizing agent is a chelating agent, such as any of(and in some embodiments selected from the group consisting of) edetate,citrate, malic acid, pentetate, tromethamine, derivatives thereof, andmixtures thereof. In some embodiments, the stabilizing agent is acitrate or a derivative thereof (such as sodium citrate). In someembodiments, the composition comprises sodium citrate and sodiumchloride. In some embodiments, the stabilizing agent is an edetate or aderivative thereof (such as EDTA). In some embodiments, the stabilizingagent is sodium gluconate. In some embodiments, the stabilizing agent issodium pyrophosphate.

In some embodiments, the composition comprises (1) particles (such asnanoparticles) comprising (in various embodiments consisting of orconsisting essentially of) a poorly water soluble pharmaceutical agentand biocompatible polymer (such as a carrier protein, which may bealbumin such as HSA); and (2) a stabilizing agent, wherein stability ofthe composition is enhanced as compared to that of a composition withoutthe stabilizing agent. In some embodiments, the composition comprisesparticles (such as nanoparticles) comprising (in various embodimentsconsisting of or consisting essentially of) (1) docetaxel andbiocompatible polymer (such as carrier protein, which may be albuminsuch as HSA); and (2) a stabilizing agent, wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, the docetaxel is coated with thebiocompatible polymer (such as carrier protein). In some embodiments,the stabilizing agent is a chelating agent, such as any of (and in someembodiments selected from the group consisting of) edetate, citrate,malic acid, pentetate, tromethamine, derivatives thereof, and mixturesthereof. In some embodiments, the stabilizing agent is a citrate or aderivative thereof (such as sodium citrate). In some embodiments, thecomposition comprises sodium citrate and sodium chloride. In someembodiments, the stabilizing agent is an edetate or a derivative thereof(such as EDTA). In some embodiments, the stabilizing agent is a malicacid. In some embodiments, the stabilizing agent is sodium gluconate. Insome embodiments, the stabilizing agent is sodium pyrophosphate.

In some embodiments, the composition comprises (1) particles (such asnanoparticles) comprising (in various embodiments consisting of orconsisting essentially of) a poorly water soluble pharmaceutical agentand albumin; and (2) a stabilizing agent, wherein the weight ratio ofthe albumin to the poorly water soluble pharmaceutical agent in thecomposition is about 0.01:1 to about 100:1, and wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, the poorly water soluble agentis coated with albumin. In some embodiments, the composition comprises(1) particles (such as nanoparticles) comprising (in various embodimentsconsisting of or consisting essentially of) a poorly water solublepharmaceutical agent and albumin; and (2) a stabilizing agent, whereinthe weight ratio of the albumin to the poorly water solublepharmaceutical agent in the composition is about 18:1 or less (includingfor example any of about 1:1 to about 18:1, about 2:1 to about 15:1,about 3:1 to about 12:1, about 4:1 to about 10:1, and about 9:1), andwherein stability of the composition is enhanced as compared to that ofa composition without the stabilizing agent. In some embodiments, thepoorly water soluble agent is coated with albumin.

In some embodiments, the composition comprises (1) particles (such asnanoparticles) comprising (in various embodiments consisting of orconsisting essentially of) docetaxel and albumin; and (2) a stabilizingagent, wherein the weight ratio of albumin and the docetaxel in thecomposition is about 0.01:1 to about 100:1, and wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent. In some embodiments, the composition comprises (1)particles (such as nanoparticles) comprising (in various embodimentsconsisting of or consisting essentially of) docetaxel and albumin; and(2) a stabilizing agent, wherein the weight ratio of albumin and thedocetaxel in the composition is about 18:1 or less (including forexample any of about 1:1 to about 18:1, about 2:1 to about 15:1, about3:1 to about 12:1, about 4:1 to about 10:1, and about 9:1), and whereinstability of the composition is enhanced as compared to that of acomposition without the stabilizing agent. In some embodiments, thedocetaxel is coated with albumin. In some embodiments, the compositionis substantially free (such as free) of surfactant. In some embodiments,the composition comprises a stable aqueous suspension of particles (suchas nanoparticles) comprising docetaxel and albumin (such as particles ofdocetaxel coated with albumin), wherein the composition furthercomprises a stabilizing agent, wherein the weight ratio of albumin andthe docetaxel in the composition is about 18:1 or less (including forexample about 1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 toabout 12:1, about 4:1 to about 10:1, and about 9:1), and whereinstability of the composition is enhanced as compared to that of acomposition without the stabilizing agent. In some embodiments, thecomposition comprises a dry (such as lyophilized) composition that canbe reconstituted, resuspended, or rehydrated to form generally a stableaqueous suspension of particles (such as nanoparticles) comprisingdocetaxel and albumin (such as docetaxel coated with albumin), whereinthe composition further comprises a stabilizing agent, wherein theweight ratio of albumin and the docetaxel in the composition is about18:1 or less (including for example any of about 1:1 to about 18:1,about 2:1 to about 15:1, about 3:1 to about 12:1, about 4:1 to about10:1, and about 9:1), and wherein stability of the composition isenhanced as compared to that of a composition without the stabilizingagent. In some embodiments, the stabilizing agent is a chelating agent,such as any of (and in some embodiments selected from the groupconsisting of) edetate, citrate, malic acid, pentetate, tromethamine,derivatives thereof, and mixtures thereof. In some embodiments, thestabilizing agent is a citrate or a derivative thereof (such as sodiumcitrate). In some embodiments, the composition comprises sodium citrateand sodium chloride. In some embodiments, the stabilizing agent is anedetate or a derivative thereof (such as EDTA). In some embodiments, thestabilizing agent is sodium gluconate. In some embodiments, thestabilizing agent is sodium pyrophosphate.

In some embodiments, the particles (such as nanoparticles) in thecomposition have an average or mean diameter of no greater than aboutany of 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 nm. In someembodiments, the average or mean diameter of the particles is betweenabout 20 to about 400 nm. In some embodiments the average or meandiameter of the particles is between about 40 to about 200 nm. In someembodiments, the particles or droplets are sterile-filterable.

The compositions described herein may be a stable aqueous suspension ofthe poorly water soluble pharmaceutical agent, such as a stable aqueoussuspension of the poorly water soluble pharmaceutical agent at aconcentration of any of about 0.1 to about 100 mg/ml, including forexample about 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about1 to about 15 mg/ml, about 1 to about 10 mg/ml, about 2 to about 8mg/ml, about 4 to about 6 mg/ml, and about 5 mg/ml. In some embodiments,the concentration of the poorly water soluble pharmaceutical agent is atleast about any of 1 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml,20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml.

In some embodiments, the composition is a dry (such as lyophilized)composition that can be reconstituted, resuspended, or rehydratedgenerally to form a stable aqueous suspension of the poorly watersoluble pharmaceutical agent. In some embodiments, the composition is aliquid (such as aqueous) composition obtained by reconstituting orresuspending a dry composition. In some embodiments, the composition isan intermediate liquid (such as aqueous) composition that can be dried(such as lyophilized).

In some embodiments, the composition is suitable for parenteral (such asintravenous) administration. In some embodiments, the composition issuitable for multidose administration. In some embodiments, thecomposition is sterile filterable. In some embodiments, the compositiondoes not cause significant side effects in an individual (such as human)when administered to the individual. In some embodiments, thecompositions described herein are substantially free (such as free) ofsurfactants. The stabilizing agent containing compositions describedherein may further comprise a sugar (including, for example, sucrose,mannitol, fructose, lactose, maltose, and trehalose) or otherlyophilization or reconstitution aids.

In some embodiments, the amount of the stabilizing agent in thecomposition is below the level that induces a toxicological effect(i.e., above a clinically acceptable level of toxicity) or is at a levelwhere a potential side effect can be controlled or tolerated when thecomposition is administered to an individual.

In another aspect, there are provided compositions (such aspharmaceutical composition) comprising docetaxel, wherein the docetaxelused for preparation of the composition is in anhydrous form (forexample the docetaxel may be anhydrous prior to being incorporated intothe composition). In some embodiments, the composition further comprisesa stabilizing agent (such as the stabilizing agents described herein).Compositions which include use of anhydrous docetaxel are furtherdescribed in a section below.

In some embodiments, the composition comprises docetaxel and abiocompatible polymer (such as a carrier protein, for example, albumin),wherein the docetaxel used for preparation of the composition is inanhydrous form. In some embodiments, the composition comprises particles(such as nanoparticles) comprising docetaxel and a biocompatible polymer(such as a carrier protein, for example albumin), wherein the docetaxelused for preparation of the composition is in anhydrous form.

In some embodiments, the composition comprises nanoparticles comprisingdocetaxel and albumin, wherein the docetaxel used for preparation of thecomposition is in anhydrous form. In some embodiments, the weight ratioof albumin and docetaxel in the composition is less than about 18:1,including for example any of about 1:1 to about 18:1, about 2:1 to about15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, about 9:1. Insome embodiments, the docetaxel is coated with albumin. In someembodiments, the nanoparticles in the composition have an average ormean particle size of no greater than about 200 nm. In some embodiments,the particles in the composition are sterile filterable. In someembodiments, the nanoparticles in the compositions have two or more ofthese properties.

In some embodiments, the composition comprises docetaxel and asurfactant, wherein the docetaxel used for preparation of thecomposition is in anhydrous form. In some embodiments, the surfactantused in preparation of the composition is anhydrous. In someembodiments, the surfactant is a polysorbate (such as Tween 80). In someembodiments, the surfactant is Cremophor. In some embodiments, thecomposition further comprises a stabilizing agent (such as thestabilizing agents described herein).

The compositions prepared with anhydrous docetaxel may be dry (such aslyophilized) compositions. In some embodiments, the composition is aliquid (such as aqueous) composition obtained by reconstituting orresuspending a dry composition. In some embodiments, the composition isan intermediate liquid (such as aqueous) composition that can be dried(such as lyophilized).

Also provided are unit dosage forms of compositions described herein,articles of manufacture comprising the inventive compositions or unitdosage forms in suitable packaging (such as vials or vessels (includingsealed vials or vessels and sterile sealed vials or vessels)), and kitscomprising the compositions. The invention also provides methods ofmaking the compositions as described herein.

Also provided are methods of stabilizing a poorly water solublepharmaceutical agent in a composition. In some embodiments, there isprovided a method of stabilizing a poorly water soluble pharmaceuticalagent in a composition (such as a nanoparticle composition), comprisingcombining the composition (such as nanoparticle composition) comprisinga poorly water soluble pharmaceutical agent with a stabilizing agent,wherein the resultant composition is stable under the same conditionunder which the composition is unstable prior to the addition of thestabilizing agent. In some embodiments, the method further comprisesidentifying and selecting a composition that is unstable under one ormore conditions. In some embodiments, the composition for selectioncomprises a poorly water soluble pharmaceutical agent and a carrierprotein (such as albumin).

Methods of using the compositions described herein are also provided.For example, in some embodiments, there is provided a method of treatingcancer in an individual (such as human), comprising administering to theindividual an effective amount of a composition comprising a poorlywater soluble antineoplastic agent, a carrier protein (such as albumin),and a stabilizing agent, wherein stability of the composition isenhanced as compared to that of a composition without the stabilizingagent. In some embodiments, there is provided a method of treatingcancer in an individual (such as human), comprising administering to theindividual an effective amount of a composition comprising docetaxel, acarrier protein (such as albumin), and a stabilizing agent, whereinstability of the composition is enhanced as compared to that of acomposition without the stabilizing agent. In some embodiments, thecomposition comprises particles (such as nanoparticles) comprisingdocetaxel and carrier protein. In some embodiments, the compositioncomprises particles (such as nanoparticles) comprising docetaxel andalbumin (such as albumin-comprising nanoparticle formulations ofdocetaxel or Nab-docetaxel). In some embodiments, the compositioncomprises Nab-docetaxel and citrate. In some embodiments, thecomposition comprises Nab-docetaxel, citrate, and sodium chloride (suchas about 200 mM sodium chloride and about 300 mM sodium citrate). Insome embodiments, the cancer is any of: prostate cancer, colon cancer,head and neck cancer, breast cancer, pancreatic cancer, lung cancer, andovarian cancer. In some embodiments, the cancer is solid tumor. In someembodiments, the composition is administered at least about any of onceevery three weeks, once every two weeks, once a week, twice a week,three times a week, four times a week, five times a week, six times aweek, or daily. In some embodiments, the composition is administered(with or without breaks) for at least about any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or more month(s). In some embodiments, the compositionis administered via any of intravenous, intraarterial, oral, topical, orinhalational routes.

General reference to “the compositions” or “compositions” includes andis applicable to compositions of the invention. The invention alsoprovides pharmaceutical compositions comprising the components describedherein.

Reference to docetaxel herein applies to docetaxel or its derivatives(or analogs) and accordingly the invention contemplates and includesboth these embodiments. Reference to “docetaxel” is to simplify thedescription and is exemplary. Derivatives or analogs of docetaxelinclude, but are not limited to, compounds that are structurally similarto docetaxel or are in the same general chemical class as docetaxel,e.g., taxanes. In some embodiments, the derivative or analog ofdocetaxel retains similar biological, pharmacological, chemical and/orphysical property (including, for example, functionality) of docetaxel.Examples of docetaxel derivatives or analogs include paclitaxel andortataxel. This same principle of description applies to other agentsprovided herein such as including, for example, stabilizing agents andpoorly water soluble pharmaceutical agents (such as taxane (includingpaclitaxel, ortataxel, or other taxanes), geldanamycin, 17-allyl aminogeldanamycin, thiocolchicine and its dimers, rapamycin, cyclosporine,epothilone, radicicol, and combretastatin).

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Stabilizing Agents

Various compositions described herein comprise a stabilizing agent.“Stabilizing agent” used herein refers to an agent that enhances thestability of the composition as compared to a composition withoutaddition of the stabilizing agent. In some embodiments, the stability ofthe stabilizing agent-containing composition is at least about 1.5×(including for example at least about any of 2×, 3×, 4×, 5×, 6, 7×, 8×,9×, 10×, 15×, 20×, 25×, 30×, or more) greater as compared to that of acomposition without the stabilizing agent.

As described above, stability of a composition can be evaluated by theability of the poorly water soluble pharmaceutical agent to remainnon-precipitated or non-sedimented (for example based on visualobservation and/or microscopic observation) in a liquid suspension overa certain period of time. Stability of a dry (such as a lyophilized)composition can be evaluated based on the behavior of the liquidsuspension resulting from reconstitution or rehydration of the drycomposition.

In some embodiments, the stabilizing agent delays or preventsprecipitation or sedimentation of the poorly water solublepharmaceutical agent in a liquid suspension. In some embodiments, thestabilizing agent delays or prevents crystallization of the poorly watersoluble pharmaceutical agent in the composition. In some embodimentswhen the composition comprises particles of poorly water soluble agents,the stabilizing agent may prevent or delay changes of particle sizes inthe composition.

The stabilizing agents are particularly useful for compositions thatwould otherwise exhibit significant instability. For example, in someembodiments, the composition prior to the addition of the stabilizingagent is stable for less than about 24 hours (including for example lessthan about any of 12, 10, 8, 6, 4, or 2 hours). In some embodiments, thepoorly water soluble pharmaceutical agent in a liquid suspension priorto the addition of the stabilizer precipitates or sediments in less thanabout 24 hours (including for example less than about any of 12, 10, 8,6, 4, or 2 hours). In some embodiments, the composition prior to theaddition of the stabilizing agent precipitates or sediments in less thanabout 24 hours when the concentration of the poorly water solublepharmaceutical agent is more than about 0.1 mg/ml (including for examplemore than about any of 0.5 mg/ml, 1 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, or 10 mg/ml). In some embodiments, thecomposition prior to the addition of the stabilizing agent precipitatesor sediments upon dilution of the composition for parentaladministration. Addition of stabilizing agent to those compositionsallows the compositions to remain stable (for example not precipitate orsediment) under similar conditions. Accordingly, in some embodiments,there is provided a composition comprising a poorly water solublepharmaceutical agent and a stabilizing agent, wherein the composition(such as a nanoparticle composition) is stable under the same conditionunder which the composition without the stabilizing agent is unstable.In some embodiments, the stabilizing agent delays or preventsprecipitation or sedimentation of the poorly water solublepharmaceutical agent in a liquid suspension of a composition under acondition where the poorly water soluble pharmaceutical agent wouldotherwise precipitate or sediment.

Suitable stabilizing agents include but are not limited to sodiumcitrate (all forms, 0.01-20% w/v), sodium pyrophosphate (0.1-10% w/v),EDTA (all forms, 0.01-20%), pentetate (all forms, 0.01-20%), sodiumgluconate (0.1-10% w/v) and suitable combinations thereof. The weightpercentage (w/v) refers to the percentage of the stabilizing agent in aliquid composition, or, in the case of a solid composition, the weightpercentage (w/v) of the stabilizing agent upon reconstitution orrehydration. The stabilizing agent should be used in an amountsufficient to increase the stability of the formulation. Preferably, theamount of the stabilizing agent used will provide a stable compositionthat does not show evidence of precipitation or sedimentation for atleast about 8 hours, more preferably at least about 24 hours afterreconstitution or rehydration, more preferably for at least about 48hours, most preferably for at least about 72 hours.

In some embodiments, the stabilizing agent is a chelating agent. Thesechelating agents are either specific to a particular metal ion (such ascalcium, zinc, magnesium, etc.), or show a broad spectrum of metal ionspecificity. In some embodiments, the chelating agent is a polydentate.In some embodiments, the chelating agent comprises one or morecarboxylic acid groups. In some embodiments, the chelating agent is notdeferoxamine. Suitable chelating agents include, but are not limited to,edetate, citrate, malic acid, pentetate, tromethamine, and derivativesthereof.

One stabilizing agent contemplated herein is an edetate, i.e.,ethylenediaminetetraacetic acid (EDTA) and derivatives thereof. Suitableedetates include disodium edetate, trisodium edetate, tetrasodiumedetate and disodium calcium edetate. In some embodiments, the edetateis present in the compositions in a concentration of about 0.01 mg/ml toabout 200 mg/ml, including for example about 0.05 mg/ml to about 150mg/ml, about 0.1 mg/ml to about 100 mg/ml, about 0.2 to about 50 mg/ml,about 0.5 mg/ml to about 20 mg/ml, about 1 mg/ml to about 10 mg/ml, andabout 1 mg/ml to about 5 mg/ml. In some embodiments, the weight ratio ofthe edetate and the poorly water soluble pharmaceutical agent (such asdocetaxel) in the composition is about 0.002:1 to about 40:1, includingfor example about 0.01:1 to about 30:1, about 0.02:1 to about 20:1,about 0.04:1 to about 10:1, about 0.1:1 to about 4:1, about 0.2:1 toabout 2:1, about 0.2:1 to about 1:1.

Another stabilizing agent contemplated herein is citrate or a derivativethereof (i.e.; citric acid or derivatives thereof), such as sodiumcitrate. Suitable concentrations of citrate include, for example, about0.1 mg/ml to about 200 mg/ml, including for example any of about 0.2mg/ml to about 100 mg/ml, about 0.3 mg/ml to about 50 mg/ml, about 0.5mg/ml to about 10 mg/ml, and about 1 mg/ml to about 5 mg/ml. In someembodiments, the concentration of citrate is less than about 200 mg/ml,such as less than about any of 100, 50, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3,2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 mg/ml. In someembodiments, the weight ratio of the citrate and the poorly watersoluble pharmaceutical agent (such as citrate) in the composition isabout 0.02:1 to about 40:1, including for example any of about 0.04:1 toabout 20:1, about 0.06:1 to about 10:1, about 0.1:1 to about 2:1, andabout 0.2:1 to about 1:1. In some embodiments, the weight ratio of thecitrate and the poorly water soluble pharmaceutical agent in thecomposition is less than about any of 20:1, 10:1, 8:1, 5:1, 2:1, 1:1,0.8:1, 0.5:1, 0.2:1, and 0.1:1.

Any form of citrate is acceptable for use in the present invention, andinclude, for example, citric acid and sodium citrate. Sodium citrate isparticularly preferred. When sodium citrate is utilized, suitableconcentrations include from about 1 to 600 mM. When citrate and sodiumchloride are utilized, suitable concentrations include from about 1 to600 mM and 1 to 1000 mM, respectively. In some embodiments, theconcentrations of citrate and sodium chloride are about 50 to about 200mM and about 300 to about 500 mM, respectively. In some embodiments, thecomposition comprises about 50 mM citrate (such as sodium citrate) andabout 500 mM sodium chloride. In some embodiments, the compositioncomprises about 200 mM citrate (such as sodium citrate) and about 300 mMsodium chloride. In some embodiments, the composition is a dry (such aslyophilized) composition, wherein the weight ratio of the citrate todocetaxel in the composition is about 17:1 and, when sodium chloride ispresent, the weight ratio of the sodium chloride to docetaxel is about3.5:1. In other embodiments, the stabilizing agent is not a citrate(i.e., other than citrate).

The stabilizing agent can also be a pentetate (including calciumtrisodium pentetate). In some embodiments, the amount of pentetate is ina concentration of about 0.01 mg/ml to about 200 mg/ml, including forexample any of about 0.05 mg/ml to about 150 mg/ml, about 0.1 mg/ml toabout 100 mg/ml, about 0.2 to about 50 mg/ml, about 0.5 mg/ml to about20 mg/ml, about 1 mg/ml to about 10 mg/ml, and about 1 mg/ml to about 5mg/ml. In some embodiments, the weight ratio of the pentetate and thepoorly water soluble pharmaceutical agent (such as docetaxel) in thecomposition is about 0.002:1 to about 40:1, including for example any ofabout 0.01:1 to about 30:1, about 0.02:1 to about 20:1, about 0.04:1 toabout 10:1, about 0.1:1 to about 4:1, about 0.2:1 to about 2:1, about0.2:1 to about 1.1.

Another stabilizing agent contemplated herein is tromethamine.Tromethamine as used herein, refers to2-amino-2-hydroxymethyl-1,3-propanediol, also known as TRIS. In someembodiments, tromethamine is in a concentration of about 0.1 mg/ml toabout 100 mg/ml, including for example about 0.5 mg/ml to about 50mg/ml, about 1 mg/ml to about 10 mg/ml, and about 2 mg/ml to about 5mg/ml. In some embodiments, the weight ratio of the tromethamine and thepoorly water soluble pharmaceutical agent in the composition is about0.02:1 to about 20:1, including for example 0.1:1 to about 10:1, about0.2:1 to about 2:1, and about 0.4:1 to about 1:1.

Other suitable metal chelating stabilizing agents and their exemplaryamount include, but are not limited to, potassium sorbate (0.5 mg/ml),sodium ascorbate (1 mg/ml), sodium formaldehyde sulfoxylate (0.1 mg/ml),and monothiolglycerol (5 mg/ml).

In some embodiments, the stabilizing agent is sodium pyrophosphate.Suitable concentration of sodium pyrophosphate include any of about 0.1to about 10% (w/v), about 0.5 to about 5%, and about 1 to about 2%. Insome embodiments, the weight ratio of the sodium pyrophosphate and thepoorly water soluble pharmaceutical agent in the composition is any ofabout 0.2:1 to about 20:1, about 1:1 to about 10:1, about 2:1 to about4:1.

In some embodiments, the stabilizing agent is sodium gluconate. Suitableconcentration of sodium gluconate include any of about 0.1 to about 10%(w/v), about 0.5 to about 5%, and about 1 to about 2%. In someembodiments, the weight ratio of the sodium gluconate and the poorlywater soluble pharmaceutical agent in the composition is any of about0.2:1 to about 20:1, about 1:1 to about 10:1, about 2:1 to about 4:1.

In some embodiments, the compositions described herein comprise at leasttwo (including for example at least any of 2, 3, 4, 5, 6, 7, 8, 9, or10) different stabilizing agents (such as stabilizing agents describedherein).

Poorly Water Soluble Pharmaceutical Agents

The compositions described herein comprise poorly water solublepharmaceutical agents. For example, the solubility in water of thepoorly water soluble agent at 20-25° C. may be less than about 10 mg/ml,including for example less than about any of 5, 2, 1, 0.5, 0.2, 0.1,0.05, 0.02, and 0.01 mg/ml.

Poorly water soluble pharmaceutical agents contemplated for use in thepractice of the present invention include poorly water solublepharmaceutically active agents, diagnostic agents, agents of nutritionalvalue and the like. Poorly water soluble pharmaceutical agents can be,for example, analgesics/antipyretics, anesthetics, antiasthmatics,antibiotics, antidepressants, antidiabetics, antifungal agents,antihypertensive agents, anti-inflammatories, antineoplastics,antianxiety agents, immunosuppressive agents, antimigraine agents,sedatives, antianginal agents, antipsychotic agents, antimanic agents,antiarrhythmics, antiarthritic agents, antigout agents, anticoagulants,thrombolytic agents, antifibrinolytic agents, hemorheologic agents,antiplatelet agents, anticonvulsants, antiparkinson agents,antihistamines/antipruritics, agents useful for calcium regulation,antibacterial agents, antiviral agents, antimicrobials, anti-infectives,bronchodialators, hormones, hypoglycemic agents, hypolipidemic agents,antiulcer/antireflux agents, antinauseants/antiemetics, oil-solublevitamins (e.g., vitamins A, D, E, K, and the like).

In some embodiments, the poorly water soluble pharmaceutical agent is anantineoplastic agent. In some embodiments, the poorly water solublepharmaceutical agent is a chemotherapeutic agent.

Suitable poorly water soluble pharmaceutical agents include, but are notlimited to, taxanes (such as paclitaxel, docetaxel, ortataxel and othertaxanes), epothilones, camptothecins, colchicines, geladanamycins,amiodarones, thyroid hormones, amphotericin, corticosteroids, propofol,melatonin, cyclosporine, rapamycin (sirolimus) and derivatives,tacrolimus, mycophenolic acids, ifosfamide, vinorelbine, vancomycin,gemcitabine, SU5416, thiotepa, bleomycin, diagnostic radiocontrastagents, and derivatives thereof. Other poorly water solublepharmaceutical agents that are useful in the inventive compositions aredescribed in, for example, U.S. Pat. Nos. 5,916,596, 6,096,331,6,749,868, and 6,537,539. Additional examples of poorly water solublepharmaceutical agents include those compounds which are poorly watersoluble and which are listed in the “Therapeutic Category and BiologicalActivity Index” of The Merck Index (12^(th) Edition, 1996).

In some embodiments, the poorly water soluble pharmaceutical agent isany of (and in some embodiments selected from the group consisting of)paclitaxel, docetaxel, ortataxel or other taxane or taxane analog,17-allyl amino geldanamycin (17-AAG), 18-derivatized geldanamycin,camptothecin, propofol, amiodarone, cyclosporine, epothilone, radicicol,combretastatin, rapamycin, amphotericin, liothyronine, epothilone,colchicine, thiocolchicine and its dimers, thyroid hormone, vasoactiveintestinal peptide, corticosteroids, melatonin, tacrolimus, mycophenolicacids, epothilones, radicicols, combretastatins, and analog orderivative thereof. In some embodiments, the poorly water solublepharmaceutical agent is any of (and in some embodiments selected fromthe group consisting of) paclitaxel, docetaxel, ortataxel or othertaxanes, geldanamycin, 17-allyl amino geldanamycin, thiocolchicine andits dimers, rapamycin, cyclosporine, epothilone, radicicol, andcombretastatin. In some embodiments, the poorly water solublepharmaceutical agent is rapamycin. In some embodiments, the poorly watersoluble pharmaceutical agent is 17-AAG. In some embodiments, the poorlywater soluble pharmaceutical agent is a thiocolchicine dimer (such asIDN5404).

In some embodiments, the poorly water soluble pharmaceutical agent is ataxane or derivative thereof, which includes, but is not limited to,paclitaxel, docetaxel and IDN5109 (ortataxel), or a derivative thereof.In some embodiments, the composition comprises a non-crystalline and/oramorphous taxane (such as paclitaxel or a derivative thereof). In someembodiments, the composition is prepared by using an anhydrous taxane(such as anhydrous docetaxel or a derivative thereof).

In some embodiments, the poorly water soluble pharmaceutical agent isdocetaxel or a derivative thereof. In some embodiments, the docetaxel inthe composition is noncrystalline or amorphous. In some embodiments, thedocetaxel is in any one or more of the following forms: anhydrate,hemihydrate, dihydrate, and trihydrate forms. Anhydrous docetaxel hasbeen shown to produce more stable formulation than those made with ahydrated docetaxel such as docetaxel trihydrate or hemi-hydrate, and isparticularly useful for the preparation of the docetaxel compositionsdescribed herein.

Biocompatible Polymers and Carrier Proteins

The compositions described herein may also comprise biocompatiblepolymers, such as carrier proteins further described herein.

As used herein, the term “biocompatible” describes a substance that doesnot appreciably alter or affect in any adverse way, the biologicalsystem into which it is introduced. Biocompatible polymer includesnaturally-occurring or synthetic biocompatible materials such asproteins, polynucleotides, polysaccharides (e.g., starch, cellulose,dextrans, alginates, chitosan, pectin, hyaluronic acid, and the like),and lipids. Suitable biocompatible polymers include, for example,naturally occurring or synthetic proteins such as albumin, insulin,hemoglobin, lysozyme, immunoglobulins, α-2-macroglobulin, fibronectin,vitronectin, fibrinogen, casein and the like, as well as combinations ofany two or more thereof. Synthetic polymers include, for example,polyalkylene glycols (e.g., linear or branched chain), polyvinylalcohol, polyacrylates, polyhydroxyethyl methacrylate, polyacrylic acid,polyethyloxazoline, polyacrylamides, polyisopropyl acrylamides,polyvinylpyrrolidone, polylactide/glycolide and the like, andcombinations thereof.

The term “proteins” refers to polypeptides or polymers of amino acids ofany length (including full length or fragments), which may be linear orbranched, comprise modified amino acids, and/or be interrupted bynon-amino acids. The term also encompasses an amino acid polymer thathas been modified naturally or by intervention; for example, disulfidebond formation, glycosylation, lipidation, acetylation, phosphorylation,or any other manipulation or modification. Also included within thisterm are, for example, polypeptides containing one or more analogs of anamino acid (including, for example, unnatural amino acids, etc.), aswell as other modifications known in the art. The proteins describedherein may be naturally-occurring, i.e., obtained or derived from anatural source (such as blood), or synthesized (such as chemicallysynthesized or by synthesized by recombinant DNA techniques).

Examples of suitable proteins include proteins normally found in bloodor plasma, which include; but are not limited to, albumin,immunoglobulin including IgA, lipoproteins, apolipoprotein B, α-acidglycoprotein, β-2-macroglobulin, thyroglobulin, transferrin,fibronectin, factor VII, factor VIII, factor IX, factor X, and the like.In some embodiments, the carrier protein is non-blood protein, such ascasein, α-lactalbumin, β-lactoglobulin. The proteins may either benatural in origin or synthetically prepared. In some embodiments, theprotein is albumin, such as HSA. HSA is a highly soluble globularprotein of M_(r) 65K and consists of 585 amino acids. HSA is the mostabundant protein in the plasma and accounts for 70-80% of the colloidosmotic pressure of human plasma. The amino acid sequence of HSAcontains a total of 17 disulphide bridges, one free thiol (Cys 34), anda single tryptophan (Trp 214). Intravenous use of HSA solution has beenindicated for the prevention and treatment of hypovolumic shock (see,e.g., Tullis, JAMA, 237, 355-360, 460-463, (1977)) and Houser et al.,Surgery, Gynecology and Obstetrics, 150, 811-816 (1980)) and inconjunction with exchange transfusion in the treatment of neonatalhyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis andHemostasis, 6, 85-120, (1980)). Other albumins are contemplated, such asbovine serum albumin. Use of such non-human albumins could beappropriate, for example, in the context of use of these compositions innon-human mammal, such as the veterinary animals (including domesticpets and agricultural animals).

Human serum albumin (HSA) has multiple hydrophobic binding sites (atotal of eight for fatty acids, an endogenous ligand of HSA) and binds adiverse set of pharmaceutical agents, especially neutral and negativelycharged hydrophobic compounds (Goodman et al., The Pharmacological Basisof Therapeutics, 9^(th) ed, McGraw-Hill New York (1996)). Two highaffinity binding sites have been proposed in subdomains IIA and IIIA ofHSA, which are highly elongated hydrophobic pockets with charged lysineand arginine residues near the surface which function as attachmentpoints for polar ligand features (see, e.g., Fehske et al., Biochem.Pharmcol., 30, 687-92 (1981), Vorum, Dan. Med. Bull., 46, 379-99 (1999),Kragh-Hansen, Dan. Med. Bull., 1441, 131-40 (1990), Curry et al., Nat.Struct. Biol., 5, 827-35 (1998), Sugio et al., Protein. Eng., 12, 439-46(1999), He et al., Nature, 358, 209-15 (1992), and Carter et al., Adv.Protein. Chem., 45, 153-203 (1994)). Paclitaxel and propofol have beenshown to bind HSA (see, e.g., Paal et al., Eur. J. Biochem., 268(7),2187-91 (2001), Purcell et al., Biochim. Biophys. Acta, 1478(1), 61-8(2000), Altmayer et al., Arzneimittelforschung, 45, 1053-6 (1995), andGamido et al., Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994)). Inaddition, docetaxel has been shown to bind to human plasma proteins(see, e.g., Urien et al., Invest. New Drugs, 14(2), 147-51 (1996)).

To provide an example, carrier proteins are further described below. Itis understood that this description generally applies to biocompatiblepolymers.

The carrier protein (such as albumin) in the composition generallyserves as a carrier for the poorly water soluble pharmaceutical agent,i.e., the carrier protein in the composition makes the poorly watersoluble pharmaceutical agent more readily suspendable in an aqueousmedium or helps maintain the suspension as compared to compositions notcomprising a carrier protein. This can avoid the use of toxic solventsfor solubilizing the poorly water soluble pharmaceutical agent, andthereby can reduce one or more side effects of administration of thepoorly water soluble pharmaceutical agent into an individual (such as ahuman). Thus, in some embodiments, the composition described herein issubstantially free (such as free) of surfactants (such as Tween 20). Acomposition is “substantially free of surfactant” if the amount ofsurfactant in the composition is not sufficient to cause one or moreside effect(s) in an individual when the composition is administered tothe individual.

In some embodiments, the carrier protein is associated with the poorlywater soluble pharmaceutical agent, i.e., the composition comprisescarrier protein-associated poorly water soluble pharmaceutical agent.“Association” or “associated” is used herein in a general sense andrefers to the carrier protein affecting the behavior and/or property ofthe poorly water soluble pharmaceutical agent in an aqueous composition.For example, the carrier protein and the poorly water solublepharmaceutical agent are considered as being “associated” if the carrierprotein makes the poorly water soluble pharmaceutical agent more readilysuspendable in an aqueous medium as compared to a composition withoutthe carrier protein. As another example, the carrier protein and thepoorly water soluble pharmaceutical agent is associated if the carrierprotein stabilizes the poorly water soluble pharmaceutical agent in anaqueous suspension. For example, the carrier protein and the poorlywater soluble pharmaceutical agent can be present in a particle or ananoparticle, which are further described herein.

A poorly water soluble pharmaceutical agent is “stabilized” by a carrierprotein in an aqueous suspension if it remains suspended in an aqueousmedium (such as without visible precipitation or sedimentation) for anextended period of time, such as for at least about any of 0.1, 0.2,0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, or 72hours. The suspension is generally, but not necessarily, suitable foradministration to an individual (such as human). As described above,stability of the suspension is in some embodiments evaluated at roomtemperature (such as 20-25° C.) or refrigerated conditions (such as 4°C.). Stability can also be evaluated under accelerated testingconditions, such as at a temperature that is higher than about 40° C. Asdescribed above, the stability of the suspension can further be enhancedby addition of the stabilizing agents described herein.

The carrier protein and the poorly water soluble pharmaceutical agent inthe composition can be associated in various manners. For example, insome embodiments, the carrier protein is in admixture with the poorlywater soluble pharmaceutical agent. In some embodiments, the carrierprotein encapsulates or entraps the poorly water soluble pharmaceuticalagent. In some embodiments, the carrier protein is bound (such asnon-covalently bound) to the poorly water soluble pharmaceutical agent.In some embodiments, the composition may exhibit one or more of theabove aspects.

In some embodiments, the composition comprises particles (such asnanoparticles) comprising (in various embodiments consisting essentiallyof) a poorly water soluble pharmaceutical agent and a carrier protein.When the poorly water soluble pharmaceutical agent is in a liquid form,the particles or nanoparticles are also referred to as droplets ornanodroplets. In some embodiments, the poorly water soluble agent iscoated with the carrier protein. Particles (such as nanoparticles) ofpoorly water soluble pharmaceutical agents have been disclosed in, forexample, U.S. Pat. Nos. 5,916,596; 6,506,405; and 6,537,579 and also inU.S. Pat. App. Pub. No. 2005/0004002A1.

In some embodiments, the composition comprises particles (such asnanoparticles) with an average or mean diameter of no greater than about1000 nanometers (nm), such as less than about any of 900, 800, 700, 600,500, 400, 300, 200, and 100 nm. In some embodiments, the average or meandiameter of the particles is no greater than about 200 nm. In someembodiments, the average or mean diameter of the particles is betweenabout 20 to about 400 nm. In some embodiments, the average or meandiameter of the particles is between about 40 to about 200 nm. In someembodiments, the nanoparticles in the composition have an average ormean particle size of no greater than about 200 nm. In some embodiments,the particles are sterile-filterable.

The particles (such as nanoparticles) described herein may be present ina dry formulation (such as lyophilized composition) or suspended in abiocompatible medium. Suitable biocompatible media include, but are notlimited to, water, buffered aqueous media, saline, buffered saline,optionally buffered solutions of amino acids, optionally bufferedsolutions of proteins, optionally buffered solutions of sugars,optionally buffered solutions of vitamins, optionally buffered solutionsof synthetic polymers, lipid-containing emulsions, and the like.

The amount of carrier protein in the composition described herein willvary depending on the poorly water soluble pharmaceutical agent andother components in the composition. In some embodiments, thecomposition comprises a carrier protein in an amount that is sufficientto stabilize the poorly water soluble pharmaceutical agent in an aqueoussuspension, for example, in the form of a stable colloidal suspension(such as a stable suspension of nanoparticles). In some embodiments, thecarrier protein is in an amount that reduces the sedimentation rate ofthe poorly water soluble pharmaceutical agent in an aqueous medium. Forparticle-containing compositions, the amount of the carrier protein alsodepend on the size and density of particles of the poorly water solublepharmaceutical agent.

In some embodiments, the carrier protein is present in an amount that issufficient to stabilize the poorly water soluble pharmaceutical agent inan aqueous suspension at a certain concentration. For example, theconcentration of the poorly water soluble pharmaceutical agent in thecomposition is about 0.1 to about 100 mg/ml, including for example anyof about 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 toabout 10 mg/ml, about 2 to about 8 mg/ml, and about 4 to about 6 mg/ml.In some embodiments, the concentration of the poorly water solublepharmaceutical agent is at least about any of 1.3 mg/ml, 1.5 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml.In some embodiments, the carrier protein is present in an amount thatavoids use of surfactants (such as Tween 80 or Cremophor), so that thecomposition is free or substantially free of surfactant (such as Tween80 or Cremophor).

In some embodiments, the composition, in liquid form, comprises fromabout 0.1% to about 50% (w/v) (e.g. about 0.5% (w/v), about 5% (w/v),about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v),about 40% (w/v), about 50% (w/v)) of the carrier protein. In someembodiments, the composition, in liquid form, comprises about 0.5% toabout 5% (w/v) of the carrier protein.

In some embodiments, the weight ratio of carrier protein, e.g., albumin,to the poorly water soluble pharmaceutical agent is such that asufficient amount of poorly water soluble pharmaceutical agent binds to,or is transported by, the cell. While the weight ratio of carrierprotein to pharmaceutical agent will have to be optimized for differentcarrier protein and drug combinations, generally the weight ratio ofcarrier protein, e.g., albumin, to pharmaceutical agent (w/w) is about0.01:1 to about 100:1, including for example any of about 0.02:1 toabout 50:1, about 0.05:1 to about 20:1, about 0.1:1 to about 20:1, about1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 12:1,about 4:1 to about 10:1, about 5:1 to about 9:1, and about 9:1. In someembodiments, the carrier protein to pharmaceutical agent weight ratio isabout any of 18:1 or less, such as about any of 15:1 or less, 14:1 orless, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 orless, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less,and 3:1 or less.

In some embodiments, the carrier protein allows the composition to beadministered to an individual (such as human) without significant sideeffects. In some embodiments, the carrier protein (such as albumin) isin an amount that is effective to reduce one or more side effects ofadministration of the poorly water soluble pharmaceutical agent to ahuman. The term “reducing one or more side effects of administration ofthe poorly water soluble pharmaceutical agent” refers to reduction,alleviation, elimination, or avoidance of one or more undesirableeffects caused by the poorly water soluble pharmaceutical agent, as wellas side effects caused by delivery vehicles (such as solvents thatrender the poorly water soluble pharmaceutical agents suitable forinjection) used to deliver the poorly water soluble pharmaceuticalagent. Such side effects include, for example, myelosuppression,neurotoxicity, hypersensitivity, inflammation, venous irritation,phlebitis, pain, skin irritation, peripheral neuropathy, neutropenicfever, anaphylactic reaction, venous thrombosis, extravasation, andcombinations thereof. These side effects, however, are merely exemplaryand other side effects, or combination of side effects, associated withvarious pharmaceutical agents can be reduced.

In some embodiments; the composition comprises particles (such asnanoparticles) comprising (in various embodiments consisting of orconsisting essentially of) a poorly water soluble pharmaceutical agentand an albumin, wherein the weight ratio of the albumin to the poorlywater soluble pharmaceutical agent in the composition (w/w) is about0.01:1 to about 100:1, including for example any of about 0.02:1 toabout 50:1, about 0.05:1 to about 20:1, about 0.1:1 to about 20:1, about1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 12:1,about 4:1 to about 10:1, about 5:1 to about 9:1, and about 9:1. In someembodiments, the carrier protein to pharmaceutical agent weight ratio isabout any of 18:1 or less, 15:1 or less, 14:1 or less, 13:1 or less.12:1 or less, 11:1 or less, 10:1 or less, 9:1 or less, 8:1 or less, 7:1or less, 6:1 or less, 5:1 or less, 4:1 or less, and 3:1 or less. In someembodiments, the poorly water soluble pharmaceutical agent is coatedwith the albumin. In some embodiments, the particles (such asnanoparticles) comprising a poorly water soluble pharmaceutical agentand albumin are suspended in an aqueous medium (such as an aqueousmedium containing albumin). For example, the composition can be acolloidal suspension of the poorly water soluble pharmaceutical agentparticles (such as nanoparticles). In some embodiments, the compositionis a dry (such as lyophilized) composition that can be reconstituted orresuspended to astable suspension of particles or described herein. Theconcentration of the poorly water soluble pharmaceutical agent in theliquid composition or reconstituted composition can be dilute (0.1mg/ml) or concentrated (100 mg/ml), including for example any of about0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 to about 10mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 to about 6 mg/ml, and 5mg/ml. In some embodiments, the concentration of the poorly watersoluble pharmaceutical agent (such as docetaxel) is greater than about0.1 mg/ml. In some embodiments, the concentration of the poorly watersoluble pharmaceutical agent is greater than about any of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, or 50 mg/ml. In some embodiments, the poorly water solublepharmaceutical agent is a taxane or a derivative thereof (such asdocetaxel or a derivative thereof).

In some embodiments, the composition comprises particles (such asnanoparticles) comprising docetaxel, such as nanoparticles with anaverage or mean diameter of between about 20 to about 400 nm. In someembodiments, the particles have an average or mean diameter of betweenabout 40 to about 200 nm. In some embodiments, the composition comprisesparticles (such as nanoparticles) comprising (in various embodimentsconsisting essentially of) docetaxel and albumin. In some embodiments,the docetaxel is coated with albumin. In some embodiments, the weightratio of the albumin to the docetaxel (w/w) in the composition is about0.01:1 to about 100:1, including for example any of about 0.02:1 toabout 50:1, about 0.05:1 to about 20:1, about 1:1 to about 18:1, about2:1 to about 15:1, about 3:1 to about 12:1. In some embodiments, thealbumin to docetaxel ratio (w/w) is about any of 18:1 or less, 15:1 orless, 14:1 or less, 13:1 or less, 12:1 or less, 10:1 or less, 9:1 orless, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less,and 3:1 or less.

In some embodiments, the particles (such as nanoparticles) comprisingdocetaxel and albumin are suspended in an aqueous medium (such as anaqueous medium containing the albumin). For example, the composition canbe a colloidal suspension of the docetaxel-containing particles (such asnanoparticles). In some embodiments, the composition is a dry (such aslyophilized composition) that can be reconstituted to an aqueoussuspension of the docetaxel-containing particles. In some embodiments,the concentration of the docetaxel in the composition is between about0.1 mg/ml and about 100 mg/ml, including for example any of about 0.1 toabout 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 to about 10 mg/ml,about 2 to about 8 mg/ml, about 4 to about 6 mg/ml, and about 5 mg/ml.In some embodiments, the concentration of docetaxel is at least aboutany of 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml.

Anhydrous Docetaxel

In addition to the use of stabilizing agents described herein (such assodium citrate and sodium citrate/sodium chloride), it has beensurprisingly found that the use of anhydrous docetaxel results in a morestable formulation than can be made with a hydrated docetaxel such asdocetaxel trihydrate or hemi-hydrate. The anhydrous docetaxelformulations of the present invention further improve the stability ofthe aqueous nanoparticle suspensions such that stability of thesuspensions, either before or after lyophilization, exceeds 1 day. Inaddition, the benefits of added stability of anhydrous docetaxel alsoextend to conventional formulations such as a formulation in Tween 80,Cremophor or other known surfactants.

Thus, in accordance with the present invention, docetaxel can bedissolved in pharmaceutically acceptable solvent or solvents at a finalconcentration in the range of about 1-99% v/v, more preferably in therange of about 5-25% v/v. Solvents include, for example, chlorinatedsolvents, ethyl acetate, ethanol, tetrahydrofuran, dioxane,acetonitrile, acetone, dimethyl sulfoxide, dimethyl formamide,methylpyrrolidinone, oils such as soybean oil, safflower oil and otherinjectable oils and the like.

In some embodiments, there is provided a composition comprisingdocetaxel, wherein the docetaxel used for preparation of the compositionis in an anhydrous form. In some embodiments, the invention provides acomposition comprising docetaxel, wherein at least some of the docetaxelin the composition is in an anhydrous form. For example, in someembodiments, at least about 10% (such as at least about any of 20%, 30%,40%, and 50%) of the docetaxel in the composition is in an anhydrousform. In some embodiments, the composition further comprises astabilizing agent (such as the stabilizing agent described herein).

In some embodiments, the composition comprises docetaxel and abiocompatible polymer (such as a carrier protein described herein, forexample albumin), wherein the docetaxel used for preparation of thecomposition is in an anhydrous form. In some embodiments, thecomposition comprises docetaxel, a biocompatible polymer (such as acarrier protein described herein, for example albumin) and a stabilizingagent (such as a stabilizing agent described herein), wherein thedocetaxel used for preparation of the composition is in an anhydrousform. In some embodiments, the composition is substantially free (suchas free) of surfactants. In some embodiments, the composition comprisessurfactant.

In some embodiments, the invention provides a composition comprisingdocetaxel and a biocompatible polymer (such as a carrier protein, forexample albumin), wherein at least some of the docetaxel in thecomposition is in an anhydrous form. For example, in some embodiments,at least about 10% (such as at least about any of 20%, 30%, 40%, and50%) of the docetaxel in the composition is in an anhydrous form. Insome embodiments, the composition further comprises a stabilizing agent(such as the stabilizing agent described herein).

In some embodiments, the invention provides a composition comprisingdocetaxel and a surfactant (such as anhydrous surfactant), wherein thedocetaxel used for preparation of the composition is in an anhydrousform. In some embodiments, the surfactant used for preparation of thecomposition is in an anhydrous form. Suitable surfactants include, forexample, polysorbate (such as Tweens) and Cremophor. In someembodiments, the composition may further comprise a stabilizing agentdescribed herein. In some embodiments, the invention provides acomposition comprising docetaxel and a surfactant, wherein at least someof the docetaxel in the composition is in an anhydrous form. Forexample, in some embodiments, at least about 10% (such as at least aboutany of 20%, 30%, 40%, and 50%) of the docetaxel in the composition is inan anhydrous form.

In some embodiments, the composition described herein is a dry (such aslyophilized) composition that can be reconstituted, resuspended, orrehydrated generally to form a stable aqueous suspension of thedocetaxel. In some embodiments, the composition is a liquid (such asaqueous) composition obtained by reconstituting or resuspending a drycomposition. In some embodiments, the composition is an intermediateliquid (such as aqueous) composition that can be dried (such aslyophilized).

In some embodiments, there is provided a method of preparingcompositions comprising docetaxel and a surfactant, wherein the methodcomprises combining an anhydrous docetaxel with the surfactant. In someembodiments, the surfactant used for preparation of the composition isanhydrous. In some embodiment, there is provided a method of preparing acomposition comprising docetaxel and a biocompatible polymer (such asthe carrier proteins, for example albumin), wherein the method comprisescombining an anhydrous docetaxel with a biocompatible polymer (such as acarrier protein, for example albumin). Also provided are compositionsproduced by methods described herein.

Other Components in the Compositions

The compositions described herein can include other agents, excipients,or stabilizers to improve properties of the composition. Examples ofsuitable excipients and diluents include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents. Examples of emulsifying agents includetocopherol esters such as tocopheryl polyethylene glycol succinate andthe like, Pluronic®, emulsifiers based on polyoxy ethylene compounds,Span 80 and related compounds and other emulsifiers known in the art andapproved for use in animals or human dosage forms. The compositions canbe formulated so as to provide rapid, sustained or delayed release ofthe active ingredient after administration to the patient by employingprocedures well known in the art.

Preferred compositions for administration by injection include thosecomprising a poorly water soluble pharmaceutical agent as the activeingredient in association with a surface-active agent (or wetting agentor surfactant), or in the form of an emulsion (e.g., as a water-in-oilor oil-in-water emulsion). Other ingredients can be added, for example,mannitol or other pharmaceutically acceptable vehicles, if necessary.

In some embodiments, the composition is suitable for administration to ahuman. In some embodiments, the composition is suitable foradministration to a mammal such as, in the veterinary context, includingdomestic pets and agricultural animals. There are a wide variety ofsuitable formulations of the inventive composition (see, e.g., U.S. Pat.Nos. 5,916,596 and 6,096,331). The following formulations and methodsare merely exemplary and are in no way limiting. Formulations suitablefor oral administration can consist of (a) liquid solutions, such as aneffective amount of the compound dissolved in diluents, such as water,saline, or orange juice, (b) capsules, sachets or tablets, eachcontaining a predetermined amount of the active ingredient, as solids orgranules, (c) suspensions in an appropriate liquid, (d) suitableemulsions, and (e) powders. Tablet forms can include one or more oflactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Injectable formulations are preferred.

Formulations suitable for aerosol administration comprise the inventivecomposition include aqueous and non-aqueous, isotonic sterile solutions,which can contain anti-oxidants, buffers, bacteriostats, and solutes, aswell as aqueous and non-aqueous sterile suspensions that can includesuspending agents, solubilizers, thickening agents, stabilizers, andpreservatives, alone or in combination with other suitable components,which can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also can be formulated as pharmaceuticalsfor non-pressured preparations, such as in a nebulizer or an atomizer.

In some embodiments, the composition is formulated to have a pH in therange of about 4.5 to about 9.0, including for example pH in the rangesof any of about 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5to about 7.0. In some embodiments, the pH of the composition isformulated to no less than about 6, including for example no less thanabout any of 6.5, 7, or 8 (such as about 7.5 or about 8). Thecomposition can also be made to be isotonic with blood by the additionof a suitable tonicity modifier, such as glycerol.

Also provided are articles of manufacture comprising the compositionsdescribed herein in suitable packaging. Suitable packaging forcompositions described herein are known in the art, and include, forexample, vials (such as sealed vials), vessels (such as sealed vessels),ampules, bottles, jars, flexible packaging (e.g., sealed Mylar orplastic bags), and the like. These articles of manufacture may furtherbe sterilized and/or sealed. Also provided are unit dosage formscomprising the compositions described herein. These unit dosage formscan be stored in a suitable packaging in single or multiple unit dosagesand may also be further sterilized and sealed.

The present invention also provides kits comprising compositions (orunit dosages forms and/or articles of manufacture) described herein andmay further comprise instruction(s) on methods of using the composition,such as uses further described herein. In some embodiments, the kit ofthe invention comprises the packaging described above. In otherembodiments, the kit of the invention comprises the packaging describedabove and a second packaging comprising a buffer. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for performing any methods describedherein.

Kits may also be provided that contain sufficient dosages of the poorlywater soluble pharmaceutical agent (such as docetaxel) as disclosedherein to provide effective treatment for an individual for an extendedperiod, such as any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months or more. Kits may also include multiple unit doses of the poorlywater soluble pharmaceutical agent and pharmaceutical compositions andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies.

Method of Making and Using the Compositions

Also provided are methods of making and using compositions describedherein. For example, there is provided a method of preparing acomposition comprising a poorly water soluble pharmaceutical agent (suchas a taxane, for example, paclitaxel, docetaxel, or ortataxel),optionally a biocompatible polymer (such as a carrier protein, forexample albumin), and a stabilizing agent, wherein stability of thecomposition is enhanced as compared to that of a composition without thestabilizing agent, comprising combining (such as admixing) a compositioncontaining a poorly water soluble pharmaceutical agent and optionally abiocompatible polymer (such as a carrier protein) with a stabilizingagent.

Also provided are methods for the formation of nanoparticles ofdocetaxel prepared under conditions of high shear forces (e.g.,sonication, high pressure homogenization or the like). The preparationof nanoparticles from biocompatible polymers (e.g., albumin) isdisclosed in, for example, U.S. Pat. Nos. 5,916,596; 6,506,405 and6,537,579 and also in U.S. Patent Publication 2005/0004002 A1incorporated herein by reference.

Briefly, the poorly water soluble pharmaceutical agent (such asdocetaxel) is dissolved in an organic solvent, and the solution can beadded to an aqueous albumin solution. The mixture is subjected to highpressure homogenization. The organic solvent can then be removed byevaporation. The dispersion obtained can be further lyophilized.Suitable organic solvents include, for example, ketones, esters, ethers,chlorinated solvents, and other solvents known in the art. For example,the organic solvent can be methylene chloride or chloroform/ethanol (forexample with a ratio of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1).

It was surprisingly found that compositions of docetaxel, such as thoseprepared in the above cited references, have stability lasting less than1 day. In fact, when tested, many of the compositions were stable foronly 4 to 8 hours. The present invention allows for increasing liquidstability and post-reconstitution stability by addition of certainstabilizers before the formation of nanoparticles or after thenanoparticles have formed.

There are therefore provided methods of stabilizing a compositioncomprising a poorly water soluble pharmaceutical agent, comprisingcombining the composition with a stabilizing agent, wherein theresultant composition is stable under the same condition under which thecomposition is unstable prior to the addition of the stabilizing agent.In some embodiments, the method further comprises identifying andselecting a composition that is unstable under certain conditions. Insome embodiments, the composition for selection comprises a poorly watersoluble pharmaceutical agent and a carrier protein (such as albumin). Insome embodiments, the composition for selection comprises particles(such as nanoparticles) comprising the poorly water solublepharmaceutical agent and a carrier protein (such as albumin).

Pharmaceutically acceptable excipients can also be added to thecomposition. The pharmaceutically acceptable excipients may be asolution, emulsion or suspension. For example, an emulsion of propofolin oil and stabilized by lecithin, is well known in the art. Otherinvention emulsion or nanoparticle formulations may also be prepared. Anemulsion is formed by homogenization under high pressure and high shearforces. Such homogenization is conveniently carried out in ahigh-pressure homogenizer, typically operated at pressures in the rangeof about 3,000 up to 30,000 psi. Preferably, such processes are carriedout at pressures in the range of about 6,000 up to 25,000 psi. Theresulting emulsion comprises very small nanodroplets of the nonaqueoussolvent containing the dissolved pharmacologically active agent and verysmall nanodroplets of the protein-stabilizing agent. Acceptable methodsof homogenization include processes imparting high shear and cavitationsuch as, for example, high-pressure homogenization, high shear mixers,sonication, high shear impellers and the like.

Colloidal systems prepared in accordance with the present invention canbe further converted into powder form by removal of the water, e.g., bylyophilization at a suitable temperature-time profile. The protein(e.g., HSA) itself acts as a cryoprotectant, and the powder is easilyreconstituted by addition of water, saline or buffer, without the needto use conventional cryoprotectants such as mannitol, sucrose, glycineand the like. While not required, it is of course understood thatconventional cryoprotectants can be added to the pharmaceuticalcompositions if so desired.

The stabilizing agent can either be admixed with the poorly watersoluble pharmaceutical agent and/or the carrier protein duringpreparation of the poorly water soluble pharmaceutical agent/carrierprotein composition, or added after the poorly water solublepharmaceutical agent/carrier protein composition is prepared. Forexample, the stabilizing agent can be present in a protein solutionprior to formation of the poorly water soluble pharmaceuticalagent/carrier protein composition. The stabilizing agent may also beadded along with an aqueous medium used to reconstitute/suspend thepoorly water soluble pharmaceutical agent/carrier protein composition oradded to an aqueous suspension of the carrier protein-associated poorlywater soluble pharmaceutical agent. In some embodiments, the stabilizingagent is admixed with the poorly water soluble pharmaceuticalagent/carrier protein composition prior to lyophilization. In someembodiments, the stabilizing agent is added as a dry component to thelyophilized pharmaceutical agent/carrier protein composition. In someembodiments when the composition comprises particles (such asnanoparticles), the stabilizing agent can be added either before orafter the particles are formed.

In some embodiments when the addition of the stabilizing agent changesthe pH of the composition, the pH in the composition are generally (butnot necessarily) adjusted to a desired pH. Exemplary pH values of thecompositions include, for example, about 5 to about 8.5. In someembodiments, the pH of the composition is adjusted to no less than about6, including for example no less than about any of 6.5, 7, or 8 (such asabout 7.5 or 8).

Also provided are methods of making pharmaceutical compositionscomprising combining any of the compositions described herein (includingthose above) with a pharmaceutically acceptable excipient.

Also provided herein are methods of using the compositions of thepresent invention. In some embodiments, there is provided a method fortreating a disease or condition that is responsive to a poorly watersoluble pharmaceutical agent comprising administering a compositioncomprising an effective amount of a poorly water soluble pharmaceuticalagent, optionally a biocompatible polymer (such as a carrier protein),and a stabilizing agent, wherein stability of the composition isenhanced as compared to that of a composition without the stabilizingagent. For example, in some embodiments, there is provided a method oftreating cancer in an individual (such as human) comprisingadministering to the individual a composition comprising an effectiveamount of a poorly water soluble antineoplastic agent (such asdocetaxel), optionally a carrier protein, and a stabilizing agent,wherein stability of the composition is enhanced as compared to that ofa composition without the stabilizing agent. In some embodiments, theamount of the stabilizing agent in the composition does not cause anytoxicological effects when the composition is administered into anindividual (such as human). In some embodiments, the invention providesa method of treating cancer in an individual (such as human) comprisingadministering to the individual an effective amount of docetaxel,wherein the docetaxel used for preparation of the composition is inanhydrous form. For example, the docetaxel may be anhydrous prior tobeing incorporated into the composition.

The term “effective amount” used herein refers to an amount of acompound or composition sufficient to treat a specified disorder,condition or disease such as ameliorate, palliate, lessen, and/or delayone or more of its symptoms. In reference to cancers or other unwantedcell proliferation, an effective amount comprises an amount sufficientto cause a tumor to shrink and/or to decrease the growth rate of thetumor (such as to suppress tumor growth). In some embodiments, aneffective amount is an amount sufficient to delay development. In someembodiments, an effective amount is an amount sufficient to preventoccurrence and/or recurrence. An effective amount can be administered inone or more administrations.

Cancers to be treated by compositions described herein (such as acomposition comprising a poorly water soluble antineoplastic agent suchas docetaxel, rapamycin, and 17-AAG) include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Examples ofcancers that can be treated by compositions described herein include,but are not limited to, squamous cell cancer, lung cancer (includingsmall cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, heptoma, breastcancer, colon cancer, melanoma, endometrical or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, liver cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, head and neckcancer, colorectal cancer, rectal cancer, soft-tissue sarcoma, Kaposi'ssarcoma, B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL), small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma,AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma,Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. In some embodiments,there is provided a method of treating metastatic cancer (that is,cancer that has metastasized from the primary tumor). In someembodiments, there is provided a method of reducing cell proliferationand/or cell migration. In some embodiments, there is provided a methodof treating hyperplasia.

In some embodiments, there are provided methods of treating cancer atadvanced stage(s). In some embodiments, there are provided methods oftreating breast cancer (which may be HER2 positive or HER2 negative),including, for example, advanced breast cancer, stage IV breast cancer,locally advanced breast cancer, and metastatic breast cancer. In someembodiments, the cancer is lung cancer, including, for example,non-small cell lung cancer (NSCLC, such as advanced NSCLC), small celllung cancer (SCLC, such as advanced SCLC), and advanced solid tumormalignancy in the lung. In some embodiments, the cancer is ovariancancer, head and neck cancer, gastric malignancies, melanoma (includingmetastatic melanoma), colorectal cancer, pancreatic cancer, and solidtumors (such as advanced solid tumors). In some embodiments, the canceris any of (and in some embodiments selected from the group consistingof) breast cancer, colorectal cancer, rectal cancer, non-small cell lungcancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer,liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, gliomas, glioblastomas, neuroblastomas, and multiplemyeloma. In some embodiments, the cancer is a solid tumor. In someembodiments, the cancer is any of (in some embodiments, selected fromthe group consisting of) prostate cancer, colon cancer, breast cancer,head and neck cancer, pancreatic cancer, lung cancer, and ovariancancer.

Individuals suitable for receiving these compositions depend on thenature of the poorly water soluble pharmaceutical agent, as well as thedisease/condition/disorder to be treated and/or prevented. Accordingly,the term individual includes any of vertebrates, mammals, and humansdepending on intended suitable use. In some embodiments, the individualis a mammal. In some embodiments, the individual is any one or more ofhuman, bovine, equine, feline, canine, rodent, or primate. In someembodiments, the individual is a human.

In another aspect, there is provided a method of treating carcinoma(such as colon carcinoma) in an individual, wherein the method comprisesadministering to the individual a composition comprising an effectiveamount of docetaxel and a carrier protein (such as albumin). In someembodiments, the composition further comprises a stabilizing agentdescribed herein, such as citrate. In some embodiments, the docetaxelused for preparation of the composition that is administered to theindividual is in an anhydrous form. The docetaxel and the carrierprotein may be present in the forms of nanoparticles (such asnanoparticles described herein).

The compositions described herein can be administered alone or incombination with other pharmaceutical agents, including poorly watersoluble pharmaceutical agents. For example, when the compositioncomprises a taxane (such as docetaxel), it can be co-administered withone or more other chemotherapeutic agents including, but are not limitedto, carboplatin, Navelbine® (vinorelbine), anthracycline (Doxil),lapatinib (GW57016), Herceptin, gemcitabine (Gemzar®), capecitabine(Xeloda®), alimta, cisplatin, 5-fluorouracil, epirubicin,cyclophosphamide, avastin, Velcade®, etc. In some embodiments, thetaxane composition is co-administered with a chemotherapeutic agentselected from the group consisting of antimetabolites (includingnucleoside analogs), platinum-based agents, alkylating agents, tyrosinekinase inhibitors, anthracycline antibiotics, vinca alkloids, proteasomeinhibitors, macrolides, and topoisomerase inhibitors. These otherpharmaceutical agents can be present in the same composition as the drug(such as taxane), or in a separate composition that is administeredsimultaneously or sequentially with the drug (such as taxane)-containingcomposition. Combination therapy methods using nanoparticle formulationsof taxane with other agents (or therapeutic methods) have been describedin International Patent Application No. PCT/US2006/006167.

The dose of the inventive composition administered to an individual(such as human) will vary with the particular composition, the method ofadministration, and the particular disease being treated. The doseshould be sufficient to effect a desirable response, such as atherapeutic or prophylactic response against a particular disease orcondition. For example, the dosage of docetaxel administered can beabout 1 to about 300 mg/m², including for example about 10 to about 300mg/m², about 30 to about 200 mg/m², and about 70 to about 150 mg/m².Typically, the dosage of docetaxel in the composition can be in therange of about 50 to about 200 mg/m² when given on a 3 week schedule, orabout 10 to about 100 mg/m² when given on a weekly schedule. Inaddition, if given in a metronomic regimen (e.g., daily or a few timesper week), the dosage may be in the range of about 1-50 mg/m².

Dosing frequency for the composition includes, but is not limited to, atleast about any of once every three weeks, once every two weeks, once aweek, twice a week, three times a week, four times a week, five times aweek, six times a week, or daily. In some embodiments, the intervalbetween each administration is less than about a week, such as less thanabout any of 6, 5, 4, 3, 2, or 1 day. In some embodiments, the intervalbetween each administration is constant. For example, the administrationcan be carried out daily, every two days, every three days, every fourdays, every five days, or weekly. In some embodiments, theadministration can be carried out twice daily, three times daily, ormore frequent.

The administration of the composition can be extended over an extendedperiod of time, such as from about a month up to about three years. Forexample, the dosing regime can be extended over a period of any of about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, and 36 months. In someembodiments, there is no break in the dosing schedule. In someembodiments, the interval between each administration is no more thanabout a week.

The compositions described herein can be administered to an individual(such as human) via various routes, including, for example, intravenous,intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, transmucosal, and transdermal. For example,the inventive composition can be administered by inhalation to treatconditions of the respiratory tract. The composition can be used totreat respiratory conditions such as pulmonary fibrosis, broncheolitisobliterans, lung cancer, bronchoalveolar carcinoma, and the like. In oneembodiment of the invention, nanoparticles (such as albuminnanoparticles) of the inventive compounds can be administered by anyacceptable route including, but not limited to, orally, intramuscularly,transdermally, intravenously, through an inhaler or other air bornedelivery systems and the like.

When preparing the composition for injection, particularly forintravenous delivery, the continuous phase preferably comprises anaqueous solution of tonicity modifiers, buffered to a pH range of about5 to about 8.5. The pH may also be below 7 or below 6. In someembodiments, the pH of the composition is no less than about 6,including for example no less than about any of 6.5, 7, or 8 (such asabout 7.5 or 8).

The nanoparticles of this invention can be enclosed in a hard or softcapsule, can be compressed into tablets, or can be incorporated withbeverages or food or otherwise incorporated into the diet. Capsules canbe formulated by mixing the nanoparticles with an inert pharmaceuticaldiluent and inserting the mixture into a hard gelatin capsule of theappropriate size. If soft capsules are desired, a slurry of thenanoparticles with an acceptable vegetable oil, light petroleum or otherinert oil can be encapsulated by machine into a gelatin capsule.

Also provided herein are methods of reducing side effects associatedwith administration of a poorly water soluble pharmaceutical agent to ahuman, comprising administering to a human a pharmaceutical compositioncomprising the poorly water soluble pharmaceutical agent, abiocompatible polymer (such as a carrier protein), and a stabilizingagent, wherein stability of the composition is enhanced as compared tothat of a composition without the stabilizing agent. For example, theinvention provides methods of reducing various side effects associatedwith administration of the poorly water soluble pharmaceutical agent,including, but not limited to, myelosuppression, neurotoxicity,hypersensitivity, inflammation, venous irritation, phlebitis, pain, skinirritation, peripheral neuropathy, neutropenic fever, anaphylacticreaction, hematologic toxicity, and cerebral or neurologic toxicity, andcombinations thereof. In some embodiments, there is provided a method ofreducing hypersensitivity reactions associated with administration ofthe poorly water soluble pharmaceutical agent, including, for example,severe skin rashes, hives, flushing, dyspnea, tachycardia, and others.

In addition, there is provided a method of enhancing stability ofcomposition comprising a poorly water soluble pharmaceutical agent andoptionally a biocompatible polymer (such as a carrier protein),comprising adding to the composition a stabilizing agent in an amountthat is effective to enhance stability of the composition. In someembodiments, there is provided a method of preparing a compositioncomprising a poorly water soluble agent (such as docetaxel), abiocompatible polymer (such as a carrier protein, for example albumin),and a stabilizing agent, comprising combining (such as admixing) thepoorly water soluble agent and the biocompatible polymer with thestabilizing agent. In some embodiments, the composition is a liquidcomposition. In some embodiments, the composition is apost-reconstitution composition.

The stabilizing agent can either be admixed with the poorly watersoluble pharmaceutical agent and/or the carrier protein duringpreparation of the poorly water soluble pharmaceutical agent/carrierprotein composition, or added along with an aqueous medium used toreconstitute the pharmaceutical/carrier protein composition.

In a further aspect of the invention is provided use of the compositionsdescribed herein in the manufacture of a medicament. Particularly, themanufacture of a medicament for use in the treatment of conditionsdescribed herein. Further, the pharmaceutical composition thereof,variously described herein, are also intended for use in the manufactureof a medicament for use in treatment of the conditions and, inaccordance with the methods, described herein, unless otherwise noted.

Those skilled in the art will recognize that several variations arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. Unless otherwise indicated, stabilities of thecompositions in the examples below are evaluated either at 25° C. or 4°C.

Example 1

This example demonstrates the instability of a preparation ofpharmaceutical compositions comprising docetaxel and albumin prepared asdescribed in U.S. Patent Publication 2005/0004002 A1.

30 mg of docetaxel was dissolved in 2 mL chloroform/ethanol. Thesolution was then added into 27.0 mL of HSA solution (3% w/v). Themixture was homogenized for 5 minutes at low RPM (Vitris homogenizermodel Tempest I.Q.) in order to form a crude emulsion, and thentransferred into a high pressure homogenizer (Avestin). Theemulsification was performed at 9000-40,000 psi. The resulting systemwas transferred into a Rotavap and solvent was rapidly removed atreduced pressure. The resulting dispersion was translucent and thetypical average diameter of the resulting particles was in the range50-220 nm (Z-average, Malvern Zetasizer). The dispersion was furtherlyophilized for 48 hours. The resulting cake was easily reconstituted tothe original dispersion by addition of sterile water or saline. Theparticle size after reconstitution was the same as beforelyophilization. When the liquid suspension prior to lyophilization wasstored, it was surprisingly found that while the suspension was stableat about 4-8 hours after preparation, at 24 hours, there was somesedimentation indicating instability. Similarly, for the lyophilizedreconstituted suspension it was surprisingly found that while thesuspension was stable at about 4-8 hours after preparation, at 24 hours,there was some sedimentation indicating instability. This instability at24 hours was not previously observed by the inventors as the monitoringperiod after initial formulation preparation and reconstitution wastypically only 4-8 hours.

Example 2

This example demonstrates the instability of docetaxel nanoparticlesprepared by sonication.

25.9 mg of docetaxel was added to a 20-mL scintillation vial anddissolved in 0.3 mL of chloroform. 4.7 mL of HSA (3.0%, w/v) was addedto the docetaxel dissolved mixture. The composition was sonicated (SonicDismembrator, model 550, Fisher Scientific Company, Pittsburgh, Pa.155275) is at 50% power for 1 min. The mixture was transferred into arotary evaporator, and chloroform-ethanol was rapidly removed at 45° C.,at reduced pressure. The diameter of the resulting docetaxel particleswas 250-300 nm (Z-average, Malvern Zetasizer). The suspensionprecipitated in less than 1 day.

Example 3

This example demonstrates the instability of docetaxel nanoparticlesprepared by sonication testing soybean oil as a stabilizer.

18.0 mg of docetaxel was added to a 20-mL scintillation vial anddissolved in 0.1 mL of chloroform-ethanol mixture. 0.05 mL of soybeanoil and 2.35 mL of HSA (5.0%, w/v) was added to the above organicsolvent. The sample was sonicated (Sonic Dismembrator, model 550, FisherScientific Company, Pittsburgh, Pa. 155275) for 2 min. The mixture istransferred into a rotary evaporator, and chloroform-ethanol is rapidlyremoved at 45° C., at reduced pressure. The diameter of the resultingdocetaxel particles was ˜270 nm (Z-average, Malvern Zetasizer). Thesuspension precipitated in less than 1 day.

Example 4

This example demonstrates the instability of docetaxel nanoparticlesprepared by sonication using an ethyl acetate-n-butyl acetate mixture.

22.7 mg of docetaxel was added to a 20-mL scintillation vial anddissolved in 1.0 mL of ethyl acetate-n-butyl acetate mixture. 2.4 mL ofHSA (5.0%, w/v) was added to the docetaxel dissolved in organic solvent.The sample was sonicated (Sonic Dismembrator, model 550, FisherScientific Company, Pittsburgh, Pa. 155275) at 50% power for 1 min. Themixture was transferred into a rotary evaporator, and ethyl acetate andn-butyl acetate are removed at reduced pressure. The compositionprecipitated within an hour.

Example 5

This example demonstrates the instability of docetaxel nanoparticlesprepared by high pressure homogenization.

49.0 mg of docetaxel was dissolved in 0.56 mL of chloroform. Thesolution was added to 9.6 mL of HSA (5%, w/v). The mixture waspre-homogenized to form a crude emulsion, and then transferred into ahigh pressure homogenizer (Avestin). The emulsification was performed at18,000-20,000 psi. The resulting system was transferred into a rotaryevaporator, and chloroform and t-butyl alcohol were removed at reducedpressure. The diameter of the resulting docetaxel particles was 160-175nm (Z-average, Malvern Zetasizer). Precipitation was observed in <1 day.Upon microscopic examination, the crystalline precipitates were seen.

Example 6

This example demonstrates the instability of docetaxel nanoparticlesprepared by high pressure homogenization using lecithin.

55.3 mg of docetaxel and 48.8 mg of egg lecithin were dissolved in 0.56mL of chloroform and t-butyl alcohol mixture. The solution was added to9.6 mL of HSA (5%, w/v). The mixture was pre-homogenized to form a crudeemulsion, and transferred into a high pressure homogenizer (Avestin).The emulsification was performed at 18,000-20,000 psi. The resultingsystem was transferred into a rotary evaporator, and chloroform andt-butyl alcohol were removed at reduced pressure. The diameter of theresulting docetaxel particles was 190-220 nm (Z-average, MalvernZetasizer). Precipitation was observed in <24 hours.

Example 7

This example demonstrates the instability of docetaxel nanoparticlesprepared by high pressure homogenization testing polylacticglycolic acid(PLGA).

56.3 mg of docetaxel and 40.8 mg of PLGA (50:50) were dissolved in 0.56mL of chloroform. The solution was added to 9.6 mL of HSA (5%, w/v). Themixture was pre-homogenized to form a crude emulsion, and transferredinto a high pressure homogenizer (Avestin). The emulsification wasperformed at 18,000-20,000 psi. The resulting system was transferredinto a rotary evaporator, and chloroform and t-butyl alcohol wereremoved at reduced pressure. The diameter of the resulting docetaxelparticles was 575 nm (Z-average, Malvern Zetasizer). Precipitation wasobserved in <24 hours.

Example 8

This example demonstrates the instability of docetaxel nanoparticlesprepared by high pressure homogenization testing benzoic acid.

50.3 mg of docetaxel and 3.0 mg of benzoic acid were dissolved in 0.56mL of chloroform and t-butyl alcohol mixture. The solution was added to10.0 mL of HSA (5%, w/v). The mixture was pre-homogenized to form acrude emulsion, and transferred into a high pressure homogenizer(Avestin). The emulsification was performed at 18,000-20,000 psi. Theresulting emulsion was transferred into a rotary evaporator, andchloroform and t-butyl alcohol were removed at reduced pressure. Thediameter of the resulting docetaxel particles was 160 nm (Z-average,Malvern Zetasizer). The composition precipitated in <24 hours.

Example 9

This example demonstrates the instability of docetaxel nanoparticlesprepared by high pressure homogenization testing cholesterol.

51.0 mg of docetaxel and 16.5 mg of cholesterol were dissolved in 0.56mL of chloroform and t-butyl alcohol mixture. The solution was added to10.0 mL of HSA (5%, w/v). The mixture was pre-homogenized to form acrude emulsion, and transferred into a high pressure homogenizer(Avestin). The emulsification was performed at 18,000-20,000 psi. Theresulting emulsion was transferred into a rotary evaporator, andchloroform and t-butyl alcohol were removed at reduced pressure.Precipitation occurred in <24 hours.

Example 10

This example demonstrates the stability of docetaxel nanoparticlesprepared by high pressure homogenization testing sodium citrate.

50.0 mg of docetaxel was dissolved in 0.56 mL of chloroform and t-butylalcohol mixture (10.2:1 (v/v)). The solution was added to 9.6 mL of HSA(5%, w/v) containing 100 mM (2.94% w/v) trisodium citrate. The mixturewas pre-homogenized to form a crude emulsion, and transferred into ahigh pressure homogenizer (Avestin). The emulsification was performed at18,000-20,000 psi. The resulting emulsion was transferred into a rotaryevaporator, and chloroform and t-butyl alcohol was removed at reducedpressure. The diameter of the resulting docetaxel particles were 150-225nm (Z-average, Malvern Zetasizer). The formulation was surprisinglystable >24 hours without observable precipitate.

Example 11

This example demonstrates the stability of docetaxel nanoparticlepreparation with citrate (3.9%, 133 mM) and sodium chloride (1.75%, 300mM).

The aqueous phase were prepared by adding HSA (5% by weight), sodiumcitrate (3.9% by weight) and sodium chloride (1.75% by weight) intowater for injection and stirred until dissolved. The organic phase wasprepared by dissolving docetaxel (7% by weight) into a solvent mixture(6% by volume) containing chloroform and ethanol and stirred untildissolved. Slowly, the organic phase was added to the aqueous phase andmixed with a rotorstator mixer. The batch size was 20 ml. The crudeemulsion was high pressure homogenized at 20,000 psi. The chloroform andethanol in the emulsion was then removed using a rotary evaporator at areduced pressure. The suspension was filtered by serial filtration (1.2μm, 0.8 μm, and 0.45 μl) and then lyophilized (FTS Tray Freeze Dryer).

The liquid suspension is homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of 165.6 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored at both 4°C. and 25° C. Surprisingly, the suspension was stable up to 3 days at 4°C. and >1 day at 25° C. The suspension did not exhibit any settling orprecipitation, and didn't change color or consistency. Furthermore, thelyophilized product appeared as a solid cake. The reconstitution of thelyophilized cake took <5 min. After reconstitution, the particles had anaverage particle size of 164.6 nm. The reconstituted suspension wasstored at 4° C. and surprisingly remained stable >1 day.

Example 12

This example demonstrates the stability of docetaxel nanoparticlepreparation with citrate (2.9%, 100 mM) and sodium chloride (1.75%, 300mM).

The aqueous phase was prepared by adding HSA (5% by weight), sodiumcitrate (2.9% by weight) and sodium chloride (1.75% by weight) intowater for injection and stirred until dissolved. The organic phase wasprepared by dissolving docetaxel (7% by weight) into a solvent mixture(6% by volume) containing chloroform and ethanol and stirred untildissolved. The organic phase was added to the aqueous phase and mixedusing a rotorstator mixer. The crude emulsion was high pressurehomogenized at 20,000 psi. The chloroform and ethanol in the emulsionwere removed using a rotary evaporator at reduced pressure. Thesuspension was filtered by serial filtration (1.2 μm, 0.8 μm, and 0.45μm) and lyophilized (FTS Tray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of 157.1 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored at both 4°C. and 25° C. Surprisingly, the suspension was stable up to 3 days at 4°C. and >1 day at 25° C. The suspension didn't exhibit any settling orcreaming, and didn't change color or consistency.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. After reconstitution, the particleshad an average particle size of 150.9 nm. The reconstituted suspensionwas stored at 4° C. and remained stable >1 day.

Example 13

This example demonstrates the stability of docetaxel nanoparticlepreparation with citrate (3.9%, 133 mM).

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (3.9% by weight) into water for injection and stirred untildissolved. The organic phase was prepared by dissolving docetaxel (5% byweight) into a solvent mixture (6% by volume) containing chloroform andethanol and stirred until dissolved. Slowly, the organic phase was addedto the aqueous phase and mixed using a rotorstator mixer. The batch sizewas 20 ml. The crude emulsion was high pressure homogenized at 20,000psi. The chloroform and ethanol in the emulsion were removed using arotary evaporator at reduced pressure. The suspension was filtered byserial filtration (1.2 μm, 0.8 μm, 0.45 μm and 0.22 μm) and thenlyophilized (FTS Tray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of 131 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. Surprisingly the nanoparticlesuspension was stable for >1 day.

Example 14

This example demonstrates the stability of docetaxel nanoparticlepreparation with citrate (11.7%, 400 mM).

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (11.7% by weight) and into water for injection and stirred untildissolved. The organic phase was prepared by dissolving docetaxel (5% byweight) into a solvent mixture (6% by volume) containing chloroform andethanol and stirred until dissolved. Slowly, the organic phase was addedto the aqueous phase and mixed using a rotorstator mixer. The batch sizewas 20 ml. The crude emulsion was high pressure homogenized at 20,000psi. The chloroform and ethanol in the emulsion were then removed usinga rotary evaporator at a reduced pressure. The suspension was filteredby serial filtration (1.2 μm, 0.8 μm, 0.45 μm and 0.22 μm) and thenlyophilized (FTS Tray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of 143.5 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored at both 4°C. and 25° C. Surprisingly, the suspension was stable up to 3 days at 4°C. and >1 day at 25° C. The suspension didn't exhibit any settling orcreaming, and didn't change color or consistency.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. After reconstitution, the particleshad an average particle size of 151.8 nm. Surprisingly, thereconstituted suspension was stored at 4° C. and remained stable >1 day.

Example 15

This example demonstrates the stability of docetaxel nanoparticlepreparation with citrate (7.7%, 200 mM).

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (7.7% by weight) and into water for injection and stirred untildissolved. The organic phase was prepared by dissolving docetaxel (5% byweight) into a solvent mixture (6% by volume) containing chloroform andethanol and stirred until dissolved. Slowly, the organic phase was addedto the aqueous phase and mixed using a rotorstator mixer. The batch sizewas 20 ml. The crude emulsion was high pressure homogenized at 20,000psi. The chloroform and ethanol in the emulsion were removed using arotary evaporator at reduced pressure. The suspension was filtered byserial filtration (1.2 μm, 0.8 μm, 0.45 μm and 0.22 μm) and thenlyophilized (FTS Tray Freeze Dryer).

The liquid suspension is homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles have anaverage size of 226.4 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored at both 4°C. and 25° C. Surprisingly, the suspension was stable up to 3 days at 4°C. and >1 day at 25° C. The suspension didn't exhibit any settling orcreaming, and didn't change color or consistency.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. After reconstitution, the particleshad an average particle size of 211.4 nm. The reconstituted suspensionwas stored at 4° C. and remained stable >1 day.

Example 16

This example demonstrates the stability of docetaxel nanoparticlepreparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (5.88%, 200 mM) and NaCl (1.75%, 300 mM) and into water forinjection and stirred until dissolved. The organic phase was prepared bydissolving docetaxel (5% by weight) into a solvent mixture (6% byvolume) containing chloroform and ethanol and stirred until dissolved.Slowly, the organic phase was added to the aqueous phase and mixed usinga rotorstator mixer. The batch size was 20 ml. The crude emulsion washigh pressure homogenized at 20,000 psi. The solvents in the emulsionwere removed using a rotary evaporator at reduced pressure. Thesuspension was filtered by serial filtration and then lyophilized (FTSTray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of <200 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored andsurprisingly, the suspension was stable without precipitates or sedimentfor >1 day.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. The reconstituted suspension wasstored and surprisingly remained stable >1 day.

Example 17

This example demonstrates the stability of a docetaxel nanoparticlepreparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (2.94%, 100 mM) and NaCl (2.9%, 500 mM) and into water forinjection and stirred until dissolved. The organic phase was prepared bydissolving docetaxel (5% by weight) into a solvent mixture (6% byvolume) containing chloroform and ethanol and stirred until dissolved.Slowly, the organic phase was added to the aqueous phase and mixed usinga rotorstator mixer. The batch size was 20 ml. The crude emulsion washigh pressure homogenized at 20,000 psi. The solvents in the emulsionwere removed using a rotary evaporator at reduced pressure. Thesuspension was filtered by serial filtration and then lyophilized (FTSTray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of <200 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored andsurprisingly, the suspension was stable without precipitates or sedimentfor >1 day.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. The reconstituted suspension wasstored and surprisingly remained stable >1 day.

Example 18

This example demonstrates the stability of a docetaxel nanoparticlepreparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (2.94%, 100 mM) and NaCl (3.5%, 600 mM) and into water forinjection and stiffed until dissolved. The organic phase was prepared bydissolving docetaxel (5% by weight) into a solvent mixture (6% byvolume) containing chloroform and ethanol and stirred until dissolved.Slowly, the organic phase was added to the aqueous phase and mixed usinga rotorstator mixer. The batch size was 20 ml. The crude emulsion washigh pressure homogenized at 20,000 psi. The solvents in the emulsionwere removed using a rotary evaporator at reduced pressure. Thesuspension was filtered by serial filtration and then lyophilized (FTSTray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of <200 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored andsurprisingly, the suspension was stable without precipitates or sedimentfor >1 day.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. The reconstituted suspension wasstored and surprisingly remained stable >1 day.

Example 19

This example demonstrates the stability of a docetaxel nanoparticlepreparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (1.47%, 50 mM) and NaCl (2.9%, 500 mM) and into water forinjection and stirred until dissolved. The organic phase was prepared bydissolving docetaxel (5% by weight) into a solvent mixture (6% byvolume) containing chloroform and ethanol and stirred until dissolved.Slowly, the organic phase was added to the aqueous phase and mixed usinga rotorstator mixer. The batch size was 20 ml. The crude emulsion washigh pressure homogenized at 20,000 psi. The solvents in the emulsionwere removed using a rotary evaporator at reduced pressure. Thesuspension was filtered by serial filtration and then lyophilized (FTSTray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of <200 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored andsurprisingly, the suspension was stable without precipitates or sedimentfor >1 day.

The lyophilized product appeared as a solid cake. The reconstitution ofthe lyophilized cake took <5 min. The reconstituted suspension wasstored and surprisingly remained stable >1 day.

Example 20

This example demonstrates the effect of anhydrous vs hydrated docetaxelnanoparticle preparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (200 mM) and NaCl (300 mM) and into water for injection andstirred until dissolved. The organic phase for three differentformulations were prepared by dissolving either anhydrous docetaxel,docetaxel trihydrate or docetaxel hemi-hydrate (partial hydrate) (5% byweight) into a solvent mixture (6% by volume) containing chloroform andethanol and stirred until dissolved. Slowly, the organic phase was addedto the aqueous phase and mixed using a rotorstator mixer. The crudeemulsion was high pressure homogenized at 20,000 psi. The solvents inthe emulsion were removed using a rotary evaporator at reduced pressure.The suspension was filtered by serial filtration and then lyophilized(FTS Tray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles have anaverage size of <200 nm. The three suspensions were stored andsurprisingly, the suspension containing the anhydrous docetaxel was themost stable without precipitates or sediment for >1 day. Both thehydrated docetaxel preparations showed precipitate or sediment in <1day. The same observation was seen for the lyophilized suspension uponreconstitution. Thus it was determined that the anhydrous form ofdocetaxel was most suitable for a nanoparticle docetaxel preparation.

Example 20A

This example provides a comparison of anhydrous docetaxel, docetaxeltrihydrate and docetaxel hemihydrate by Differential Scanningcalorimetry (DSC).

The three different types of docetaxel were subject to DSC usingstandard techniques. All showed a melting endotherm at about 162-166° C.However only the 2 hydrated materials showed a water dehydrationendotherm between about 74-80° C.

Example 20B

This example provides a comparison of anhydrous docetaxel, docetaxeltrihydrate and docetaxel hemihydrate by X-Ray Powder Diffraction (XRD).

The three different types of docetaxel were subject to XRD usingstandard techniques. The three materials all showed a variety of sharppeaks indicating crystallinity. However, the anhydrous material showed adifferent spectrum as compared to the two hydrated materials. Inparticular was a peak occurring at 2-theta of 7-8 for the anhydroussample which was absent from the hydrated material. This indicated adifferent crystal structure for the anhydrous docetaxel versus thehydrated docetaxels.

Example 21

This example demonstrates that the degree of hydration affects thesolubility of docetaxel and provides a comparison of the solubility ofanhydrous docetaxel, docetaxel trihydrate and docetaxel hemihydrate.

To compare if the different types of docetaxel material had differentsolubility profiles as a result of their different structures, theirsolubility rates were compared in the solvent acetonitrile. Acetonitrilewas added to a fixed amount of docetaxel from different suppliers toobtain a concentration of 5 mg/mL (anhydrous basis). The rate at whichdissolution of the different docetaxels occurred was observed. It wasobserved that the anhydrous docetaxels (from 2 different suppliers)dissolved completely in less than 1 minute. In contrast the hydratedmaterials (trihydrate and partial hydrate from 2 different suppliers)did not readily dissolve and additional solvent had to be added to afinal concentration of 2.5 mg/mL. Under these further dilutedconditions, the time to dissolve was between 5 and 10 minutes for thehydrated materials. A similar observation was made when using thesolvent chloroform. Thus, it is surprisingly found that the degree ofhydration or anhydrous nature can substantially affect the solubility ofdocetaxel.

Example 22

This example demonstrates that the degree of hydration of docetaxelaffects the stability and provides a comparison of the formulations ofdocetaxel trihydrate, hemihydrate and anhydrous docetaxel in Tween 80.

It is well known that docetaxel is formulated with Tween 80 as asolubilizer or emulsifier for the commercial product Taxotere. Thedifferent docetaxels were dissolved in Tween 80 at a concentration of 40mg/mL (on anhydrous basis). 2 mL of these solutions were observed forstability over time. It was surprisingly found that after a few days, asediment or precipitate was observed for the hydrated docetaxel but noprecipitate was observed with the anhydrous docetaxel. Thus, theanhydrous docetaxel is preferred in the Tween formulation. In additionit may be useful to use a Tween 80 or equivalent surfactant that isanhydrous or very low in water content as the anhydrous form ofdocetaxel may absorb water to form the hydrated form which could resultin precipitation.

Example 23

This example demonstrates the stability of a docetaxel nanoparticlepreparation with anhydrous docetaxel and without added stabilizers.

The aqueous phase was prepared by adding HSA (5% by weight) to water forinjection. The organic phase was prepared by dissolving anhydrousdocetaxel (5% by weight) into a solvent mixture (6% by volume)containing chloroform and ethanol and stirred until dissolved. Slowly,the organic phase was added to the aqueous phase and mixed using arotorstator mixer. The crude emulsion was high pressure homogenized at20,000 psi. The solvents in the emulsion were removed using a rotaryevaporator at reduced pressure. The suspension was filtered by serialfiltration and then lyophilized (FTS Tray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of <200 nm. The sample was also examined by microscopy andmost of the particles were <0.5 μm. The suspension was stored andsurprisingly, the suspension was stable without precipitates or sedimentfor approximately 1 day. Thus, in the absence of stabilizersNab-docetaxel prepared with anhydrous docetaxel appears to be morestable than when prepared with a hydrated form of docetaxel for whichstability is much less than 1 day, typically only a few hours.

Example 24

This example demonstrates the preparation of anhydrous docetaxelnanoparticle preparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (8.5% by weight) and sodiumcitrate (200 mM) and NaCl (300 mM) and into water for injection andstirred until dissolved. The organic was prepared by dissolvinganhydrous docetaxel (133 mg/ml) into a solvent mixture containingchloroform and ethanol (1:1) and stirring until dissolved. Slowly, theorganic phase (6% by volume) was added to the aqueous phase and mixedusing a rotorstator mixer. The batch size was 200 ml. The crude emulsionwas high pressure homogenized at 20,000 psi. The solvents in theemulsion were removed using a rotary evaporator at reduced pressure. Thesuspension was filtered by serial filtration and then lyophilized (FTSTray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of <200 nm. The suspension was stored and surprisingly,showed no precipitates or sediment for >1 day. The same observation wasseen for the lyophilized suspension upon reconstitution.

Example 25

This example demonstrates the preparation of anhydrous docetaxelnanoparticle preparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (200 mM) and NaCl (300 mM) and into water for injection andstirred until dissolved. The organic phase was prepared by dissolvinganhydrous docetaxel (160 mg/ml) into a solvent mixture containingchloroform and ethanol (1:1) and stirring until dissolved. Slowly, theorganic phase (8% by volume) was added to the aqueous phase and mixedusing a rotorstator mixer. The batch size was 200 ml. The crude emilsionwas subject to high pressure homogenization at 20,000 psi. The solventsin the emulsion was removed using a rotary evaporator at reducedpressure. The suspension was filtered by serial filtration and thenlyophilized (FTS Tray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of less than 200 nm. The suspension was stored and,surprisingly, showed no precipitation or sedimentation for more than oneday. The same observation was seen for the lyophilized suspension uponreconstitution.

Example 26

This example demonstrates the preparation of anhydrous docetaxelnanoparticle preparation with citrate/NaCl.

The aqueous phase was prepared by adding HSA (5% by weight) and sodiumcitrate (200 mM) and NaCl (300 mM) and into water for injection andstirred until dissolved. The organic phase was prepared by dissolvinganhydrous docetaxel (160 mg/ml) into a solvent mixture containingchloroform and ethanol (1:1) and stirring until dissolved. Slowly, theorganic phase (8% by volume) was added to the aqueous phase and mixedusing a rotorstator mixture. The batch size was 200 ml. The crudeemulsion was subject to high pressure homogenization at 20,000 psi. Thesolvents in the emulsion were removed using a rotary evaporator atreduced pressure. Additional albumin was added to the evaporatedsuspension to increase the albumin: drug ratio to 8:1 by weight. Thesuspension was filtered by serial filtration and then lyophilized (FTSTray Freeze Dryer).

The liquid suspension was homogeneous and off-white. Particle sizeanalysis was performed using a Malvern Zetasizer. The particles had anaverage size of less than 200 nm. The suspension was stored and,surprisingly, showed no precipitates or sediment for more than one day.The same observation was seen for lyophilized suspension uponreconstitution.

Example 27

This example demonstrates effect of pH on stability of the nanoparticlesuspension as well as on chemical degradation of docetaxel.

Formulations of nanoparticle docetaxel were prepared as described in theabove examples. The effect of pH on these formulations was testedbetween pH 4 and pH 9. Increasing pH above pH 6 was found to increasephysical stability measured in terms of nanoparticle size andsedimentation of the formulation while at the same time increasing theamount of degradation of docetaxel to 7-epi docetaxel at roomtemperature. An optimal pH range in which both the physical stabilityand chemical stability was acceptable was thus found to be between6-8.5. A more preferable pH range was 6.5-8 and a most preferable rangewas found to be pH 7.25 to 7.75.

Example 28

This example compares the stability of Nab-docetaxel prepared witheither hydrated forms of docetaxel or anhydrous docetaxel in thepresence or absence of suitable stabilizers.

Stability of these preparations was examined visually prior tolyophilization and well as upon reconstitution of the lyophilizedpreparations. In addition, the stability of lyophilized preparations(containing stabilizers) upon reconstitution was evaluated at differentconcentration of docetaxel in the reconstituted suspension. The resultsare set forth in Tables 1-3 below.

TABLE 1 Stability evaluation of Nab-docetaxel nanoparticle suspensionprior to lyophilization Stability Observations State of StabilityObservations Nab-docetaxel with Hydration of Nab-docetaxel with nostabilizer (Citrate Docetaxel stabilizers 200 mM/NaCl 300 mM)Hemihydrate Immediate sedimentation Data not obtained (Batch I) (<15min) of nanoparticle suspension once formed Trihydrate Sedimentation ofnanoparticle Sedimentation of (Batch I) suspension in approximately 1nanoparticle suspension by hour day 1 at 4° C. Anhydrous Sedimentationof nanoparticle No sedimentation of (Batch I) filtrate by day 1 at 4° C.nanoparticle filtrate for 2 days at 4° C. Anhydrous Sedimentation ofnanoparticle No sedimentation of (Batch II) filtrate by day 1 at 4° C.nanoparticle filtrate for 2 days at 4° C.

As shown in Table 1, stability of Nab-docetaxel prepared using anhydrousdocetaxel was significantly better than Nab-docetaxel prepared usinghydrated forms of docetaxel whether or not stabilizers were present inthe formulation.

Addition of stabilizers (200 mM citrate/300 mM NaCl) significantlyimproved stability of Nab-docetaxel preparations containing nostabilizer.

Addition of stabilizers improved stability of Nab-docetaxel preparationsmade from docetaxel trihydrate.

TABLE 2 Reconstitution stability of Nab-docetaxel lyophilized powdercontaining stabilizer (citrate 200 mM/NaCl 300 mM) reconstituted to 5mg/mL docetaxel in water for injection Docetaxel type:Trihydrate/Anhydrous 15 min 1 hr 2 hr 4 hr 6 hr 24 hr Trihydrate (25°C.) 1 2 2 3 4 4 Anhydrous (25° C.) 1 1 1 1 1 1 Trihydrate (40° C.) 1 2 44 4 4 Anhydrous (40° C.) 1 1 1 1 1 1 Code: 1 - No sedimentation 2 -Slight sedimentation 3 - More sedimentation 4 - Thick sedimentation 5 -Complete sedimentation

TABLE 3 Reconstitution stability of Nab-docetaxel lyophilized powdercontaining stabilizer (citrate 200 mM/NaCl 300 mM) reconstituted to 1mg/mL docetaxel in water for injection Trihydrate/Anhydrous 15 min 1 hr2 hr 4 hr 6 hr 24 hr Trihydrate (4° C.) 2 3 5 5 5 5 Anhydrous (4° C.) 13 5 5 5 5 Trihydrate (25° C.) 2 3 5 5 5 5 Anhydrous (25° C.) 1 3 5 5 5 5Trihydrate (40° C.) 3 3 5 5 5 5 Anhydrous (40° C.) 2 3 5 5 5 5 Code: 1 -No sedimentation 2 - Slight sedimentation 3 - More sedimentation 4 -Thick sedimentation 5 - Complete sedimentation

As shown in Tables 2 and 3, stability of reconstituted Nab-docetaxelcontaining stabilizers is significantly improved at a higherconcentration of 5 mg/ml docetaxel versus a lower concentration of about1 mg/ml docetaxel. Stability of the reconstituted Nab-docetaxelformulation containing stabilizers prepared with anhydrous docetaxel issignificantly better than Nab-docetaxel prepared with docetaxeltrihydrate.

Example 29

This example demonstrates the toxicity profiles of nanoparticle albuminformulation of docetaxel (Nab-docetaxel) vs Taxotere.

Maximum tolerated dose (MTD) for Nab-docetaxel and Taxotere® (Tween80-docetaxel) were determined during a dose escalation study in nudemice. Nude mice, group size of 10 per group, were treated withincreasing dose of Taxotere (0 mg/kg, 7 mg/kg, 15 mg/kg, 22 mg/kg, 33mg/kg, and 50 mg/kg) using a q4dx3 schedule. Nude mice, group size of 6per group, were treated with increasing dose of Nab-docetaxel (0 mg/kg,15 mg/kg, 22 mg/kg, 33 mg/kg, 50 mg/kg, and 75 mg/kg) using a q4dx3schedule. Animals were weighed every other day. Maximum body weight losswas plotted versus dose and fitted using a Hill equation. MTD defined asweight loss equal to 20% was calculated using the fitted data. The MTDwas 2.3 fold higher for Nab-docetaxel versus Taxotere (Tween 80docetaxel). MTDs were 47.2 mg/kg and 20.6 mg/kg for Nab-docetaxel andTaxotere, respectively.

Example 30

This example demonstrates the antitumor efficacy of inventionnanoparticle docetaxel (Nab-docetaxel) with stabilizer vs Taxotere.

Efficacy of Nab-docetaxel (prepared with 200 mM citrate and 300 mM NaCl)was compared against Taxotere in a xenograft tumor model in nude micebearing human TACT-116 colon tumor. 10 mice per group were used for thestudy. Taxotere was dosed at 15 mg/kg and Nab-docetaxel was dosed at 22mg/kg, both on a q4dx3 schedule. Nab-docetaxel (22 mg/kg) was moreeffective in tumor suppression than Taxotere (15 mg/kg, MTD) withp<0.0001. In addition, Nab-Docetaxel exhibited greater therapeutic indexthan Taxotere as maximum weight loss in the Taxotere group was 20%,while that for Nab-docetaxel was about 17%, despite a 50% higher dose.

Example 31

This example demonstrates an infusion study of Nab-docetaxel (200 mMcitrate/300 mM NaCl).

A study in rats was conducted with 5 min infusion of Nab-docetaxel, withincreasing infusion rates of Nab-docetaxel formulation containing approx200 mM of citrate/300 mM NaCl. A 5 min infusion in rats may beconsidered equivalent to 30 min infusion in humans.

Maximum safe infusion rate was ˜0.5 ml/min. This is equivalent to 0.23mmol/kg/min or 68 mg/kg/min of citrate for 5 min infusion in rats.Translated to human dose, this was equivalent to approximately 170 mgdocetaxel/m² in a 30 min infusion.

Example 32

This example demonstrates the blood biocompatibility of 5 mg/mlNab-docetaxel (200 mM citrate/300 mM NaCl).

An in vitro hemolysis study in rat blood was conducted using a placeboformulation (all components except docetaxel) and the Nab-docetaxelformulation. The placebo did not cause hemolysis even at the highest ratblood:placebo ratio of 1:1. The Nab-docetaxel formulation interferedwith the absorption reading due to the characteristic light-scatteringby nanoparticles, but when appropriate background/controls wereperformed, no hemolysis was detected at the highest ratblood:Nab-docetaxel ratio of 1:1. This demonstrates that Nab-docetaxelwith stabilizer as indicated is compatible with rat blood.

Example 33

This example provides a pilot multiple dose escalation study in rats.All Nab-docetaxel formulations described herein contain 200 mM citrateand 300 mM NaCl.

To compare the safety of the Nab-docetaxel formulation with the Taxotereformulation, rats were dosed with either Tween 80 docetaxel (sameformulation as Taxotere) or Nab-docetaxel at 5.0, 10.0, 15.0, 30.0, and50 mg/kg using a 10 minute infusion through indwelling jugular catheterson days 0, 4, and 8 for a total of three treatments. Saline (1ml/kg/min) was used as a control.

Each animal was observed and weighed daily during days 0-25. Body weightwas recorded daily for each treated animal. Signs of clinical distresswere recorded daily. Blood was collected on days 13, 16, and day 25 intoEDTA-treated tubes and subjected to differential analysis. Necropsy wasconducted on day 25.

The result of the study is shown in Table 4. As shown in the table,animals in all dose groups tolerated the first treatment with no acuteor infusion related toxicities even at the highest dose of 50 mg/kg.However, only the animals in the lowest dose level of 5 mg/kg and thecontrol saline survived until the end of the experiment (all threetreatments). All animals receiving higher doses, died either between thesecond and third doses or following the third dose.

TABLE 4 Weight loss and Mortality in dose escalation study MaximumGroups % wt. loss Mortality Observations Tween 80-docetaxel 50 mg/kg N/A3/3 dead between 2nd and 3rd dose 30 mg/kg N/A 3/3 dead between 2nd and3rd dose 15 mg/kg N/A 3/3 dead between 2nd and 3rd dose 10 mg/kg N/A 2/3dead between 2nd and 3rd dose; 1/3 after 3rd dose  5 mg/kg 24% 0/3 deadNab-docetaxel 50 mg/kg N/A 3/3 dead between 2nd and 3rd dose 30 mg/kgN/A 3/3 dead between 2nd and 3rd dose 15 mg/kg N/A 3/3 dead between 2ndand 3rd dose 10 mg/kg N/A 2/3 dead between 2nd and 3rd dose, 1/3 after3rd dose  5 mg/kg 15% 0/3 dead Control Saline  3% 0/3 dead

Weight loss of the treated animals is shown in FIG. 1. Neutropeniacomparison at 5 mg/kg docetaxel dose for Nab-docetaxel and Tween80-docetaxel is shown in FIG. 2. Weight loss for the Nab-docetaxel 5mg/kg surviving group was significantly less than that of the 5 mg/kgTween-docetaxel group (p=0.02+, ANOVA). This was parallel bysignificantly higher severe neuropenia for Tween-docetaxel (5 mg/kg)versus Nab-docetaxel (5 mg/kg), p<0.0001, ANOVA, FIG. 2) on day 13.Necropsy at the end of the experiment (day 25) in the surviving 5 mg/kggroups revealed abnormalities in ⅔ animals in the Tween-docetaxel group(one case of milky fluid accumulation in the thoracic cavity and onecase of abnormal spleen adhering to the abdominal wall, stomach, andpancreas). Nab-docetaxel (5 mg/kg) and saline animals were normal.

This pilot study showed significant improvement in safety forNab-docetaxel in terms of overall body weight loss. Neutropenia wassignificantly higher for Tween-docetaxel.

Example 34

This example demonstrates the blood kinetics of Nab-docetaxel. AllNab-docetaxel formulations described herein contain 200 mM citrate and300 mM NaCl.

Rats were divided into six groups (3 per group). On day 1, each animalwas weighed and administered a single intravenous dose of theappropriate article show below:

Group A: Taxotere, 10 mg/kg

Group B: Taxotere, 20 mg/kg

Group C: Taxotere, 30 mg/kg

Group D: Nab-docetaxel, 10 mg/kg

Group E: Nab-docetaxel, 20 mg/kg

Group F: Nab-docetaxel, 30 mg/kg

The test articles were administered over a 10+/−1 minute infusionperiod. Blood samples (200 μL) were collected from the tail vein of eachrat at the following intervals: Prior to infusion (baseline); duringinfusion (5 minutes into infusion, t=−5 minutes); and at completion ofinfusion (t=0). Blood was also collected at the following time pointsafter completion of infusion: 5, 10, and 20 minutes; 40±3 minutes; 2hours±5 minutes; 3 hours±10 minutes; 4 hours±10 minutes; 8 hours±10minutes; 24±1 hours; 48±1 hours; and 120±2 hours. Blood samples werecollected in green top (sodium heparin) tubes and processed forcollection of plasma by centrifuging at approximately 2,000 rpm forapproximately 10 minutes. Plasma samples were stored frozen untilshipped on dry ice to ALTA Analytical (El Dorado Hills, Calif.) forLC/MS analysis of docetaxel levels.

The results of the experiments are shown in FIG. 3 and Table 5. Therewere no significant differences between the PK profiles of Nab-docetaxelversus Taxotere at 10 mg/kg. However, the differences betweenNab-docetaxel and Taxotere were significant at 20 mg/kg with Cmax andAUC, 53% and 70% of Taxotere respectively and Vz and Vss were 177% and243% of Taxotere respectively. At 30 mg/kg, once again the differenceswere significant with Cmax and AUC for Nab-docetaxel 46% and 47% ofTaxotere respectively and Vz and Vss were 225% and 375% of Taxotererespectively.

TABLE 5 PK Parameters for Nab-docetaxel and Taxotere PK ParametersNab-docetaxel Taxotere p-value DOSE: 10 mg/kg HL (hr) 8.3 + 0.3 9.0 +3.2 ns Tmax (hr) 0.11 + 0.05 0.14 + 0.05 ns Cmax (ng/ml) 4,330 + 358  4953 + 1,014 ns AUC inf (hr*ng/ml) 1,588 + 77   2,069 + 615  ns Vz(L/kg) 76 + 6  63 + 14 ns Cl (L/hr/kg) 6.3 + 0.3 5.1 + 1.5 ns Vss (L/kg)28 + 1  26 + 9  ns DOSE: 20 mg/kg HL (hr) 6.3 + 1.8  5.1 + 0.31 ns Tmax(hr) 0.11 + 0.05 0.14 + 0.05 ns Cmax (ng/ml) 8,546 + 1,545 16,167 +2,804  0.01 AUC inf (hr*ng/ml) 3,953 + 419  5,664 + 500  0.01 Vz (L/kg)46 + 10 26 + 1  0.02 Cl (L/hr/kg) 5.1 + 0.5 3.5 + 0.3 0.01 Vss (L/kg)17 + 4  7 + 1 0.01 DOSE: 30 mg/kg HL (hr) 7.3 + 1.0 6.9 + 2.9 ns Tmax(hr) 0.17 + 0.00 0.14 + 0.05 ns Cmax (ng/ml) 15,800 + 5,408  34,467 +14,221 0.1 AUC inf (hr*ng/ml) 7,049 + 896  14,881 + 1,169  0.0008 Vz(L/kg) 45 + 10 20 + 8  0.03 Cl (L/hr/kg) 4.3 + 0.5 2.0 + 0.2 0.002 Vss(L/kg) 15 + 4  4 + 1 0.01

When AUC was plotted versus dose, the nonlinearity for Taxotere wasclearly evident, with Nab-docetaxel AUC being linear with respect todose (FIG. 3D). This can be explained by the micelle forming property ofTween 80, the high solubility of docetaxel in the hydrophobic micellecore and corresponding sequestration of docetaxel in the plasma (6).Furthermore, the rapid tissue distribution for Nab-docetaxel may also beexplained by utilization of the albumin/caveolae mediated transcytosisvia endothelial cells, a process previously described for Abraxane(Nab-paclitaxel).

The PK data suggests that Tween 80 in Taxotere exhibited sequestrationof docetaxel in plasma similar to that seen with Cremophor EL in thecase of Taxol. This resulted in higher Cmax and AUC and lower volumes ofdistribution for Taxotere than for Nab-docetaxel. The PK ofNab-docetaxel is linear while that for Tween 80-docetaxel (Taxotere) isnon-linear with respect to dose. The dosages described herein, i.e., 10mg/kg, 20 mg/kg, and 30 mg/kg, are equivalent to a human dosage of about60 mg/m², about 120 mg/m², and about 180 mg/m². Typically, the linearrange of PK of the Nab-docetaxel is about 10-180 mg/m².

Example 35

This example demonstrates the inhibition of drug-albumin interaction bysurfactants such as Tween 80. The experiment was done using afluorescent-labelled paclitaxel (Flutax) as a surrogate forpaclitaxel/docetaxel. Flutax was shown to have similar binding toalbumin as paclitaxel.

HSA was immobilized on 96 well plastic microplate. The immobilizedalbumin was reacted for 1 hr with constant concentration of Flutax andincreasing concentration of solvents (Cremophor EL/EtOH, Tween 80, andTPGS). The unbound ligands were washed off with buffer. Bound ligandswere quantitated using a fluorometer. The 1050 was determined using anexponential decay equation.

The results of the experiment were shown in FIG. 4. As shown in FIG. 4,albumin-paclitaxel interaction was inhibited by solvent commonly used inthe formulation of water insoluble drug such as Cremophor EL/EtOH, Tween80, and TPGS (1050 of 0.009%, 0.003%, and 0.008%, respectively).Complete inhibition occurred at 0.02% or 0.2 μl Tween 80/ml. This isclinically relevant as Taxotere treated patients exhibited 0.07-0.41 μlof Tween 80/ml of blood at the end of drug infusion.

This experiment demonstrates that Tween 80 in the Taxotere formulationmay inhibit binding of docetaxel to albumin and prevent its endothelialtranscytosis via the gp60/caveolar mechanism. The PK data in the abovestudies also support this observation.

Example 36

This example provides evaluation of antitumor activity of Nab-docetaxelagainst H29 colon carcinoma xenograft in athymic nude mice. The micewere divided into the control group and the Nab-docetaxel group (N=4mice per group, each with bilateral tumors). All Nab-docetaxelformulations described herein contain 200 mM citrate and 300 mM NaCl.

Briefly, H29 tumors were implanted subcutaneously in athymic nude mice,allowed to grow to 100 mm³ and then treated with either the control (nodrug) or Nab-docetaxel (15 mg/kg, q4dx3, iv bolus). Tumor size and bodyweight measurements were obtained three times weekly and plotted in FIG.5.

As shown in FIG. 5, there was significant inhibition of HT29 tumor invivo, p<0.0001 vs control, ANOVA. At the 15 mg/kg dose of Nab-docetaxel,mean weight loss between 10-20% suggesting that this dose may be closeto the MTD for Nab-docetaxel. The MTD for Taxotere has been reported tobe 15 mg/kg on this schedule.

Example 37

This example compares the antitumor activity of Nab-docetaxel andTaxotere using the HCT116 colon carcinoma xenograft in athymic nude micewith a 50% higher dose of Nab-docetaxel as compared to Taxotere. Themice were divided into the control group, the Nab-docetaxel group, andthe Taxotere group (N=10 mice per group). All Nab-docetaxel formulationsdescribed herein contain 200 mM citrate and 300 mM NaCl.

Briefly, antitumor activity of Nab-docetaxel and Taxotere were comparedat doses of 22 mg/kg q4×3 and 15 mg/kg q4×3, respectively in the HCT116colon carcinoma xenograft. The results of the experiments are shown inFIG. 6.

As shown in FIG. 6, both Nab-docetaxel and Taxotere showed tumorinhibition with respect to the control. As shown below tumor inhibitionwas improved with Nab-docetaxel versus Taxotere (p=0.03, ANOVA) andweight loss was somewhat lower but not statistically significant (p=ns,ANOVA) between the two groups.

In this pilot study, the antitumor activity of Nab-docetaxel wassuperior to that of Taxotere. The mice tolerated 50% higher docetaxeldose for Nab-docetaxel with somewhat lower overall body weight losscompared to Taxotere.

Example 38

This example compares the toxicity of Nab-docetaxel preparation withstabilizers (citrate/NaCl) vs Taxotere (Tween-docetaxel) in rats given asingle dose of each preparation.

Male Sprague-Dawley rats (160-180 g, n=3/group) were infused withTaxotere, or Nab-docetaxel (citrate/NaCl) Infusion time was 10 minutesand the following dose levels of docetaxel were used: 25, 50, 75, 100,and 125 mg/kg. The animals were weighed and monitored daily for signs oftoxicity/mortality. Percent mortality (%) at 7 days following treatmentwere shown in Table 6.

TABLE 6 Percent mortality in rats treated by Taxotere and Nab-docetaxel.Dose (mg/kg) 125 100 75 50 25 Taxotere 100% 100% 100% 100% 100%Nab-docetaxel  66%  66% 100%  33%  0% (+citrate)

As shown in Table 6, the Nab-docetaxel formulation were significantlyless toxic than Taxotere (Tween-docetaxel). This effect was particularlypronounced at doses of 25 and 50 mg/kg. The LD50 was calculated to be 63mg/kg for Nab-docetaxel versus approximately 12.5 mg/kg forTween-docetaxel.

Example 39

This example shows the efficacy of Nab-docetaxel in treatment ofprostate cancer in a PC3 prostate xenograft tumor model.

PC3 tumor were implanted subcutaneously in athymic nude mice, allowed togrow to 100 mm³ and then treated q4×3, i.v. with either the saline orNab-docetaxel (10, 15, 20, or 30 mg/kg) or Tween-docetaxel (10 mg/kg).Six mice in each group were evaluated.

The results of the study are shown in FIG. 7. All six Tween-docetaxeltreated mice died over the course of the study. By contrast,Nab-docetaxel was well tolerated at all dose levels. There was only onedeath at 15 mg/kg, and none was observed at the higher dose levels of 20mg/kg and 30 mg/kg. Tumor suppression was observed at all dose levels ofNab-docetaxel. In particular, at 30 mg/kg dose, there were six out ofsix complete regressions.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

1-36. (canceled)
 37. A stable docetaxel pharmaceutical injectablecomposition comprising a) docetaxel; b) polysorbate 80; and c) astabilizing agent, wherein the stabilizing agent is citric acid.
 38. Thestable docetaxel pharmaceutical injectable composition of claim 37,wherein the composition is sterile.
 39. The stable docetaxelpharmaceutical injectable composition of claim 37, wherein thecomposition is chemically stable upon prolonged storage.
 40. The stabledocetaxel pharmaceutical injectable composition of claim 37, wherein thecomposition is stable for at least about 72 hours.
 41. The stabledocetaxel pharmaceutical injectable composition of claim 37, wherein theconcentration of docetaxel is about 30 mg/ml to about 40 mg/ml.
 42. Amethod of preparing the docetaxel pharmaceutical injectable compositionof claim 37 comprising combining the stabilizing agent, docetaxel, andpolysorbate
 80. 43. The method of claim 42, wherein the method comprisesadding the stabilizing agent to a composition comprising docetaxel andpolysorbate 80.